Biomarkers Related to Interleukin-33 (IL-33)-Mediated Diseases and Uses Thereof

ABSTRACT

The present invention relates to the identification of certain biomarkers for use in identifying patients who have, or are likely to develop an IL-33 mediated disease or disorder and who are more likely to respond to therapy with an IL-33 antagonist. The invention also relates to methods of treatment of an IL-33-mediated disease or disorder in a patient by administering an IL-33 antagonist to the patient in need thereof and monitoring the effectiveness of therapy using the biomarkers described herein. Also provided are methods for decreasing the level of at least one biomarker in a subject suffering from an IL-33-mediated disease or disorder, and methods for treating such diseases or disorders according to the expression levels of one or more biomarkers. The methods of the present invention comprise administering to a subject in need thereof a pharmaceutical composition comprising an interleukin-33 antagonist.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C §119(e) of U.S.provisional application No. 62/237,624, filed Oct. 6, 2015, which isherein specifically incorporated by reference in its entirety.

REFERENCE TO A SEQUENCE LISTING

This application incorporates by reference the Sequence Listingsubmitted in Computer Readable Form as file 10187US01-Sequence.txt,created on Oct. 4, 2016 and containing 159,912 bytes.

FIELD OF THE INVENTION

The present invention relates to the identification of particularbiomarkers in a mammal that correlate with expression of IL-33 and theuse of these biomarkers to identify patients who have, or are likely todevelop, an IL-33 mediated disease or disorder and who are more likelyto respond to therapy with an IL-33 antagonist. The invention alsorelates to methods of treatment of an IL-33-mediated disease or disorderin a patient by administering an IL-33 antagonist to the patient in needthereof and monitoring the effectiveness of therapy using the biomarkersdescribed herein.

BACKGROUND

Interleukin-33 (IL-33), which is a member of the IL-1 superfamily ofcytokines, is expressed predominantly by stromal cells, such asepithelial and endothelial cells, following pro-inflammatorystimulation. IL-33 is a ligand for ST2, a toll-like/interleukin-1receptor super-family member that associates with an accessory protein,IL-1 RAcP (for reviews, see, e.g., Kakkar and Lee, Nature Reviews-DrugDiscovery 7(10):827-840 (2008), Schmitz et. al., Immunity 23:479-490(2005); Liew et. al., Nature Reviews-Immunology 10:103-110 (2010); US2010/0260770; US 2009/0041718). Upon activation of ST2/IL-1 RAcP byIL-33, a signaling cascade is triggered through downstream moleculessuch as MyD88 (myeloid differentiation factor 88) and TRAF6 (TNFreceptor associated factor 6), leading to activation of NFκB (nuclearfactor-κB), among others.

IL-33 signaling has been implicated as a factor in a variety of diseasesand disorders (Liew et. al., Nature Reviews-Immunology 10:103-110(2010)). For example, while IL-33 is protective against helminthinfection in a host and also reduces atherosclerosis by promotingT_(H)2-type immune responses, it can also promote the pathogenesis ofasthma by expanding T_(H)2 cells and mediate joint inflammation, atopicdermatitis and anaphylaxis by mast cell activation. As such, IL-33 maybe a new target for therapeutic intervention across a range of diseases;for example, blockade of IL-33 signaling may offer the potential toameliorate multiple pathogenic features of certain inflammatorydiseases.

IL-33 antagonists are described in U.S. Pat. Nos. 5,576,191; 7,666,622;8,119,771; 8,187,596; 9,090,694; 9,212,227; 9,382,318; U.S. patentpublication numbers 2007/0042978; 2009/0041718; 2010/0260770;2012/0207752; 2012/0263709; 2014/0140954; 2014/0271642; 2014/0212412; EPpatents or patent application numbers 1725261 B1; 2069784A1; 2152740A1;2283860A2; 2475388A1; and PCT publication numbers WO05/079844;WO08/132709; WO08/144610; WO09/053098; WO11/031600; WO14/152195 andWO14/164959.

Calcitonin (CT) is a polypeptide hormone, thought to be producedprimarily by parafollicular cells of the thyroid (Foster, G. V., et.al., (1964), Nature 202: 1303-1305). CT is a product of the CALCA gene,located on chromosome 11. The CALCA gene encodes the polypeptidesprocalcitonin (PCT) and PCT gene-related peptide α (proCGRPα), which aredifferentially expressed by alternative splicing (Christ-Crain, M. et.al. (2008), Crit Care Med 36:1684-1687; Hoff, A O, et. al. (2002), J.Clin. Invest. 110:1849-1857). Calcitonin (CT) is derived from a largerprecursor, Procalcitonin (PCT, 116 amino acids), which is cleaved toimmature calcitonin (33 amino acids) and then to mature calcitonin, amonomer of a 3.5-kd peptide composed of 32 amino acids. CT ispredominantly produced by neuroendocrine cells such as C cells of thethyroid and pulmonary neuroendocrine cells (PNECs) by proteolyticcleavage in the secretory granules of producing cells before beingreleased as mature CT polypeptide. Other cells that express the calcagene include mast cells, dorsal route ganglion cells (DRGs) and cells ofthe spinal cord. Multiple forms of circulating calcitonin precursors arefound in the serum of healthy and diseased individuals (Becker K. et.al. (2004), J Clin Endocrinol Metab 89:1512-1525).

Calcitonin gene related peptide (CGRP) is a member of the calcitoninfamily of peptides. α-CGRP is a 37-amino acid peptide formed from thealternative splicing of the calcitonin/CGRP gene located on chromosome11 (Amara, S G, et. al., (1982), Nature, 298 (5871): 240-244).

CT acts to reduce blood calcium, inhibits both osteoclasts and boneresorption, opposing the activity of parathyroid hormone (PTH) (Naot, D.and J. Cornish (2008), Bone 43(5): 813-818). In vivo and in vitroexperiments suggest that the major physiological function of CT is tocombat hypercalcemia in states of calcium stress such as duringpregnancy, growth or lactation (Hoff, A. O., et. al., (2002), J ClinInvest 110(12): 1849-1857; Zaidi, M., et. al., (2002), J Clin Invest110(12): 1769-1771). Diagnostically, CT is used as biomarker formedullary thyroid carcinoma (MTC). Normal circulating levels of CTpeptides are low, however under physiological conditions these levelsincrease either systemically or locally. High CT levels indicate thepresence of MTC and are used to evaluate the efficacy of surgicalextirpation and recurrences. CT is also elevated in C-cell hyperplasia,pulmonary and pancreatic tumors, kidney failures and thyroid autoimmunedisease (Becker, K. L., et. al., (2004). J Clin Endocrinol Metab 89(4):1512-1525).

To date, no biomarkers for identifying patients that have, or are proneto develop an IL-33 mediated disease or disorder, or to determine apatient's likelihood to respond to therapy with an IL-33 antagonist havebeen identified. Although IL-33 antagonists have been identified thatshow promise in the treatment of inflammatory conditions, or allergies,biomarkers that may predict the efficacy of anti-IL-33 therapy areneeded for the effective identification and selection of patientsub-populations that respond favorably to anti-IL-33 therapy.Accordingly, an unmet need exists in the art for identifying andvalidating predictive and prognostic biomarkers in patients withinflammatory conditions, or allergies, who are administered anti-IL-33therapy.

BRIEF SUMMARY OF THE INVENTION

According to certain aspects of the present invention, methods areprovided for treating, preventing and/or reducing the severity orduration of symptoms of an interleukin-33 (IL-33)-mediated disease ordisorder in a subject. The methods of the present invention compriseadministering to a subject in need thereof a pharmaceutical compositioncomprising a therapeutically effective amount of an interleukin-33(IL-33) antagonist.

In one embodiment of the present invention, the IL-33 antagonist is anantibody or antigen-binding fragment thereof that specifically bindsIL-33.

In one embodiment of the present invention, the IL-33 antagonist is areceptor-based antagonist of IL-33, such as an IL-33 trap, as describedherein.

The present invention also provides for the identification of particularbiomarkers that correlate with the expression of IL-33 in a subjectsuffering from an IL-33 mediated disease or disorder. Exemplarybiomarkers associated with an IL-33-mediated disease or disorder thatcan be evaluated and/or measured in the context of the present inventioninclude, but are not limited to, polypeptides encoded by the CALCA gene,including calcitonin, procalcitonin, and calcitonin gene-related peptide(CGRP). In one embodiment, the biomarker associated with anIL-33-mediated disease or disorder that can be evaluated and/or measuredin the context of the present invention is calcitonin. Other biomarkersassociated with an IL-33-mediated disease or disorder that can beevaluated and/or measured in the context of the present inventioninclude, but are not limited to resistin-like alpha (RETNA); chemokine(C-C motif) ligand 8 (Ccl8); serum amyloid A 3 (Saa3); Gm1975(BC117090); killer cell lectin-like receptor (Kirg1); stefin A1 (Csta);membrane-spanning 4-domain (Ms4a8a); chemokine (C-C motif) ligand 11(Ccl11); and serine (or cysteine) peptides (Serpina3f).

In a related aspect, the invention provides for the expression of atleast one biomarker in the serum of a subject suffering from an IL-33mediated disease or disorder, which correlates with the level ofexpression of IL-33 in at least one tissue sample from the subject.

In one embodiment, the serum biomarker that correlates with IL-33expression levels in at least one tissue sample is calcitonin.

In a certain embodiment, the serum biomarker that correlates with IL-33expression levels in at least one tissue sample is procalcitonin.

In a related embodiment, the serum biomarker that correlates with IL-33expression levels in at least one tissue sample is calcitoningene-related peptide (CGRP).

In another related aspect, the present invention provides a method fortreating an interleukin-33 (IL-33) mediated disease or disorder in asubject, the method comprising: (a) selecting a subject who exhibits anelevated level of at least one biomarker associated with an IL-33mediated disease or disorder prior to, or at the time of treatment, and(b) administering to the subject a pharmaceutical composition comprisinga therapeutically effective amount of an interleukin-33 antagonist.

According to a related aspect of the present invention, methods fortreating an IL-33-mediated disease or disorder are provided whichcomprise administering to a subject a pharmaceutical compositioncomprising a therapeutically effective amount of an IL-33 antagonist,wherein administration of the pharmaceutical composition to the subjectresults in a decrease in at least one biomarker associated with anIL-33-mediated disease or disorder in the subject.

In one embodiment, the biomarker that is elevated in a subject sufferingfrom an IL-33 mediated disease or disorder is calcitonin and the IL-33antagonist is a monoclonal antibody that specifically binds to IL-33.

The present invention also provides methods for decreasing the level ofone or more IL-33-mediated disease or disorder-associated biomarker(s)in a subject, which results in improving one or more IL-33-mediateddisease or disorder-associated parameter(s) in a subject, wherein themethods comprise administering to a subject in need thereof a singleinitial dose of a pharmaceutical composition comprising an IL-33antagonist. In certain embodiments, the invention provides administeringone or more secondary doses of the pharmaceutical composition comprisingthe IL-33 antagonist, wherein the administering results in furtherreduction in the level of one or more of the biomarkers described hereinand further improvement in one or more IL-33 mediated disease ordisorder-associated parameters in a subject. In one embodiment, there isa strong correlation between the level of the biomarker present in theserum of the subject having an IL-33 mediated disease or disorder andthe severity of the disease or disorder-associated parameter, e.g.infiltration of eosinophils or neutrophils into the lungs of thesubject, or an increase in cytokine levels (e.g. IL-5) in the lungs ofthe subject. In certain embodiments, the level of IL-33 in the tissue,e.g. the lung, correlates with the severity of the disease ordisorder-associated parameter. In one embodiment, the IL-33 antagonistis a human monoclonal antibody that binds specifically to IL-33. In oneembodiment, the IL-33 antagonist is an IL-33 trap, as described herein.

In a related aspect, the present invention provides methods for treatingan IL-33-mediated disease or disorder in a subject comprisingadministering to the subject a pharmaceutical composition comprising atherapeutically effective amount of an antibody or antigen-bindingfragment thereof that specifically binds IL-33, wherein the subject hasbeen diagnosed with an IL-33-mediated disease or disorder and has alsobeen selected for treatment on the basis of the subject exhibiting anelevated level of at least one IL-33-mediated disease ordisorder-associated biomarker before treatment, as compared to areference level of the biomarker (e.g., expression of the biomarker in asubset of subjects diagnosed with an IL-33-mediated disease or disorderand/or expression of the biomarker in healthy subjects).

In another related aspect, the present invention also provides methodsfor treating an IL-33-mediated disease or disorder by administering tothe subject a pharmaceutical composition comprising a therapeuticallyeffective amount of an antibody or antigen-binding fragment thereof thatspecifically binds IL-33, wherein the subject has been diagnosed with anIL-33-mediated disease or disorder, has already been treated with theanti-IL33 antagonist for a defined period of time, and has been selectedfor further treatment with the IL-33 antagonist on the basis ofexhibiting expression of at least one biomarker (e.g., calcitonin,procalcitonin, or calcitonin gene-related peptide (CGRP)) aftertreatment for a defined period of time, wherein the expression of thebiomarker is determined based on a comparison to the level of expressionof the respective biomarker in the subject prior to treatment with theanti-IL-33 antagonist.

The invention also provides a method for identifying a subject sufferingfrom an IL-33 mediated disease or disorder who is likely to respondfavorably to therapy with an IL-33 antagonist, the method comprisingobtaining a sample from the subject and measuring the level ofcalcitonin in the sample; wherein an elevated level of calcitonin, ascompared to the lower level of calcitonin in subjects not suffering froman IL-33 mediated disease or disorder, identifies the patient as apatient who is likely to respond favorably to therapy with an IL-33antagonist.

In certain embodiments, the subject who is likely to respond favorablyto therapy with an IL-33 antagonist may be identified by measuring thelevel of procalcitonin, or calcitonin gene-related peptide (CGRP) in atissue sample, wherein an elevated level of either procalcitonin, orcalcitonin gene-related peptide (CGRP), as compared to the lower levelof procalcitonin, or CGRP in subjects not suffering from an IL-33mediated disease or disorder, identifies that patient as a patient whois likely to respond favorably to therapy with an IL-33 antagonist.

In another related aspect, the invention provides a method ofdetermining whether a subject is likely to have, or likely to develop anIL-33 mediated disease or disorder, the method comprising obtaining asample from the subject and measuring in the sample the level ofcalcitonin; wherein an elevated level of calcitonin, as compared to areference standard, identifies the patient as a patient who is likely tohave, or develop an IL-33 mediated disease or disorder.

In certain embodiments, the invention provides a method of determiningwhether a subject is likely to have, or likely to develop an IL-33mediated disease or disorder, the method comprising obtaining a samplefrom the subject and measuring in the sample the level of procalcitonin,or CGRP; wherein an elevated level of procalcitonin, or CGRP, ascompared to reference standards for either of the two biomarkers,identifies the patient as a patient who is likely to have, or develop anIL-33 mediated disease or disorder. Methods for measuring the levels ofany of the above biomarkers are known to those of skill in the art.

The sample from the subject may be a solid tissue sample, a cell sample,or a blood sample. The solid tissue sample may be selected from thegroup consisting of heart, kidney, liver, lung, colon and spleen. Thecell sample may be a dorsal root ganglion cell sample, sputum cellsample, bronchoalveolar lavage cell sample, nasal polyps cell sample,fecal cell sample, lung, colon heart, kidney, skin biopsy, or spleenbiopsy cell sample. The blood sample may be whole blood, plasma, orserum.

In one embodiment, the IL-33 antagonist to be used in the methods of theinvention is an isolated human monoclonal antibody or antigen-bindingfragment thereof that specifically binds human interleukin-33 (IL-33),wherein the antibody or antigen-binding fragment comprises: (a) thecomplementarity determining regions (CDRs) of a heavy chain variableregion (HCVR) having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162,178, 194, 210, 226, 242, 258, 274, 290, and 308; and (b) the CDRs of alight chain variable region (LCVR) having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74,90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, and316.

In one embodiment, the IL-33 antagonist to be used in the methods of theinvention is an isolated human monoclonal antibody or antigen-bindingfragment thereof that specifically binds human interleukin-33 (IL-33),wherein the anti-IL-33 antibody or antigen-binding fragment thereofcomprises the heavy and light chain CDRs of a HCVR/LCVR amino acidsequence pair selected from the group consisting of: SEQ ID NOs: 2/10,18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154,162/170, 178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/282,290/298, and 308/316.

In one embodiment, the IL-33 antagonist to be used in the methods of theinvention is an isolated human monoclonal antibody or antigen-bindingfragment thereof that specifically binds human interleukin-33 (IL-33),wherein the antibody or antigen-binding fragment comprisesHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively, selected fromthe group consisting of: SEQ ID NOs: 4-6-8-12-14-16; 20-22-24-28-30-32;36-38-40-44-46-48; 52-54-56-60-62-64; 68-70-72-76-78-80;84-86-88-92-94-96; 100-102-104-108-110-112; 116-118-120-124-126-128;132-134-136-140-142-144; 148-150-152-156-158-160;164-166-168-172-174-176; 180-182-184-188-190-192;196-198-200-204-206-208; 212-214-216-220-222-224;228-230-232-236-238-240; 244-246-248-252-254-256;260-262-264-268-270-272; 276-278-280-284-286-288;292-294-296-300-302-304; and 310-312-314-318-320-322.

In one embodiment, the IL-33 antagonist to be used in the methods of theinvention is an isolated human monoclonal antibody or antigen-bindingfragment thereof that specifically binds human interleukin-33 (IL-33),wherein the antibody or antigen-binding fragment comprises: (a) a heavychain variable region (HCVR) having an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130,146, 162, 178, 194, 210, 226, 242, 258, 274, 290, and 308; and (b) alight chain variable region (LCVR) having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74,90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, and316.

In one embodiment, the IL-33 antagonist to be used in the methods of theinvention is an isolated IL-33 receptor based antagonist (e.g. an IL-33trap), comprising a first IL-33 binding domain (D1) attached to amultimerizing domain (M), wherein D1 comprises an IL-33-binding portionof an ST2 protein. In one embodiment, the IL-33 trap further comprisesone or more additional IL-33 binding domains selected from the groupconsisting of D2, D3 and D4.

In one embodiment, the IL-33 receptor based antagonist comprises anIL-33-binding portion of an ST2 protein, an extracellular domain of anIL-1 RAcP protein, or other IL-33 binding domain.

In one embodiment, the IL-33 receptor based antagonist is an IL-33 trapcomprising the amino acid sequence selected from the group consisting ofSEQ ID NOs: 323, 324, 325, 326 and 335.

In one embodiment, the methods of the invention provide administrationof an effective amount of a second therapeutic agent useful fordiminishing at least one symptom of an IL-33 mediated disease ordisorder.

In one embodiment, the second therapeutic agent is selected from thegroup consisting of a non-steroidal anti-inflammatory (NSAID), acorticosteroid, a bronchial dilator, an antihistamine, epinephrine, adecongestant, a thymic stromal lymphopoietin (TSLP) antagonist, an IL-13antagonist, an IL-4 antagonist, an IL-4/IL-13 dual antagonist, an IL-5antagonist, an IL-6 antagonist, an IL-12/23 antagonist, an IL-22antagonist, an IL-25 antagonist, an IL-17 antagonist, an IL-31antagonist, an oral PDE4 inhibitor and another IL-33 antagonist or adifferent antibody or receptor based antagonist to IL-33.

In one embodiment, the invention provides for treatment of a subjectsuffering from an IL-33 mediated disease or disorder with an IL-33antagonist selected from an anti-IL-33 antibody or an IL-33 trap, whichmay be used in combination with any one or more of the secondtherapeutic agents noted above, wherein the effectiveness of treatmentwith these agents may be monitored by assessing the level of at leastone biomarker selected from the group consisting of calcitonin (Calca),procalcitonin, calcitonin gene-related peptide (CGRP), resistin-likealpha (RETNA), chemokine (C-C motif) ligand 8 (Ccl8), serum amyloid A 3(Saa3), Gm 1975 (BC117090), killer cell lectin-like receptor (Kirg1),stefin A1 (Csta), membrane-spanning 4-domain (Ms4a8a), chemokine (C-Cmotif) ligand 11 (Ccl11), and serine (or cysteine) peptides (Serpina3f).

In one embodiment, the biomarker is calcitonin and the calcitonin levelincreases in the serum of the subject having an IL-33 mediated diseaseor disorder, and the increase in serum calcitonin correlates with anincreased level of calcitonin and IL-33 in the lungs of the subject.

In one embodiment, the increased level of calcitonin in the serum of thesubject having an IL-33 mediated disease or disorder is reduced to areference range level following treatment with an anti-IL-33 antagonist.

In one embodiment, a subject suffering from an IL-33 mediated disease ordisorder is selected for treatment with an IL-33 antagonist on the basisof exhibiting a mean serum calcitonin level of greater than about 5pg/ml, greater than about 10 pg/ml, greater than about 50 pg/ml, orgreater than about 100 pg/ml prior to, or at the time of treatment.

In one embodiment, a subject suffering from an IL-33 mediated disease ordisorder is selected for treatment with an IL-33 antagonist on the basisof exhibiting a mean serum procalcitonin level of greater than about0.15 ng/ml, greater than about 1.0 ng/ml, greater than about 5 ng/ml,greater than about 10 ng/ml, greater than about 50 ng/ml, or greaterthan about 100 ng/ml prior to, or at the time of treatment.

In one embodiment, a subject suffering from an IL-33 mediated disease ordisorder is selected for treatment with an IL-33 antagonist on the basisof exhibiting a mean serum calcitonin gene-related peptide (CGRP) levelof greater than about 45 pg/ml, greater than about 100 pg/ml, greaterthan about 250 pg/ml, or greater than about 500 pg/ml prior to, or atthe time of treatment.

In certain embodiments, the methods of the invention provide fortreatment of an IL-33 mediated disease or disorder, which is aninflammatory disease or disorder selected from the group consisting ofasthma, allergy, allergic rhinitis, allergic airway inflammation, atopicdermatitis (AD), chronic obstructive pulmonary disease (COPD),inflammatory bowel disease (IBD), multiple sclerosis, arthritis,psoriasis, eosinophilic esophagitis, eosinophilic pneumonia,eosinophilic psoriasis, hypereosinophilic syndrome, graft-versus-hostdisease, uveitis, cardiovascular disease, pain, multiple sclerosis,lupus, vasculitis, chronic idiopathic urticaria and EosinophilicGranulomatosis with Polyangiitis (Churg-Strauss Syndrome).

In one embodiment, the IL-33 mediated disease or disorder to be treatedis an allergy and the subject selected for treatment with an IL-33antagonist is selected on the basis of an increase in calcitonin levelsthat are above normal (e.g. based on a reference level of the biomarkeras determined by established values for healthy subjects compared tosubjects diagnosed with an IL-33 mediated disease or disorder), whereinthe treatment with an IL-33 antagonist comprises administration of apharmaceutical composition comprising a therapeutically effective amountof a monoclonal antibody that specifically binds IL-33 and comprises anHCVR/LCVR amino acid sequence pair of SEQ ID NOs: 274/282, and whereinthe administration results in an improvement in at least one symptomassociated with the IL-33 disease or disorder, or an improvement in atleast one disease associated parameter; and wherein the calcitonin levelis restored to a normal range as determined by comparison to a referencestandard.

In one embodiment, the IL-33 mediated disease or disorder to be treatedis an allergy and the subject selected for treatment with an IL-33antagonist is selected on the basis of an increase in serum calcitoninlevels as determined by comparison with a reference standard, whereinthe calcitonin level correlates with an increase in IL-33 level in thesubject, or with an increase in at least one disease parameter in thesubject, and wherein the increase in IL-33 level is determined using abinding assay comprising an antibody that binds specifically to humanIL-33 and comprises a HCVR/LCVR amino acid sequence pair of SEQ ID NOs:274/282.

The present invention also includes any combination of the embodimentsdiscussed above or herein. Particular embodiments of the presentinvention will become apparent from a review of the ensuing detaileddescription.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B: Show mouse serum IL-33 (FIG. 1A) and mouse Calcitonin(CT) levels (FIG. 1B) in mouse IL-33 HDD experiment.

FIG. 2: Shows mouse Calcitonin (Calca) gene expression in the chronicHDM model.

FIG. 3: Shows the correlation between serum and lung Calcitonin (CT)levels in the chronic HDM model.

FIG. 4: Shows the correlation of serum Calcitonin (CT) and lung IL-33levels in the chronic HDM model.

FIGS. 5A, 5B and 5C: Show the parameters affected by anti-IL-33treatment in chronic HDM model. FIG. 5A: Frequency of lung neutrophils;FIG. 5B: Frequency of lung eosinophils; FIG. 5C: lung IL-5 levels.

FIGS. 6A, 6B and 6C: Show the correlation of serum Calcitonin (CT) andthe parameters affected by anti-IL-33 treatment in the chronic HDMmodel. FIG. 6A: correlation between serum CT levels and frequency oflung neutrophils; FIG. 6B: correlation between serum CT levels andfrequency of lung eosinophils; FIG. 6C: correlation between serum CT andlung IL-5 protein levels.

FIGS. 7A, 7B and 7C: Show the correlation of lung IL-33 levels and theparameters affected by anti-IL-33 treatment in the chronic HDM model.FIG. 7A: correlation between lung IL-33 levels and frequency of lungneutrophils; FIG. 7B: correlation between lung IL-33 levels andfrequency of lung eosinophils; FIG. 7C: correlation between lung IL-33and lung IL-5 protein levels.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.). Asused herein, the terms “treat”, “treating”, or the like, mean toalleviate symptoms, eliminate the causation of symptoms either on atemporary or permanent basis, or to prevent or slow the appearance ofsymptoms of the named disorder or condition.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice of the present invention,the preferred methods and materials are now described. All patents,applications and non-patent publications mentioned in this specificationare incorporated herein by reference in their entireties.

Biomarkers Associated with an IL-33-Mediated Disease or Disorder

The present invention includes methods involving the use,quantification, and analysis of biomarkers associated with anIL-33-mediated disease or disorder.

An “IL-33 mediated disease or disorder” may be selected from anyinflammatory disease or disorder such as, but not limited to, asthma,allergy, allergic rhinitis, allergic airway inflammation, atopicdermatitis (AD), chronic obstructive pulmonary disease (COPD),inflammatory bowel disease (IBD), multiple sclerosis, arthritis,psoriasis, eosinophilic esophagitis, eosinophilic pneumonia,eosinophilic psoriasis, hypereosinophilic syndrome, graft-versus-hostdisease, uveitis, cardiovascular disease, pain, multiple sclerosis,lupus, vasculitis, chronic idiopathic urticaria and EosinophilicGranulomatosis with Polyangiitis (Churg-Strauss Syndrome).

The asthma may be selected from the group consisting of allergic asthma,non-allergic asthma, severe refractory asthma, asthma exacerbations,viral-induced asthma or viral-induced asthma exacerbations, steroidresistant asthma, steroid sensitive asthma, eosinophilic asthma ornon-eosinophilic asthma and other related disorders characterized byairway inflammation or airway hyperresponsiveness (AHR).

The COPD may be a disease or disorder associated in part with, or causedby, cigarette smoke, air pollution, occupational chemicals, allergy orairway hyperresponsiveness.

The allergy may be associated with foods, pollen, mold, dust mites,animals, or animal dander.

The IBD may be selected from the group consisting of ulcerative colitis,Crohn's Disease, collagenous colitis, lymphocytic colitis, ischemiccolitis, diversion colitis, Behcet's syndrome, infective colitis,indeterminate colitis, and other disorders characterized by inflammationof the mucosal layer of the large intestine or colon.

The arthritis may be selected from the group consisting ofosteoarthritis, rheumatoid arthritis and psoriatic arthritis.

As used herein, the term “an IL-33-mediated disease ordisorder-associated biomarker”, or a “biomarker associated with anIL-33-mediated disease or disorder” means any biological response, celltype, parameter, protein, polypeptide, enzyme, enzyme activity,metabolite, nucleic acid, carbohydrate, or other biomolecule which ispresent or detectable in a patient suffering from an IL-33-mediateddisease or disorder at a level or amount that is different from (e.g.,greater than or less than) the level or amount of the marker present ordetectable in a non-IL-33-mediated disease or disorder patient.Exemplary IL-33-mediated disease or disorder-associated biomarkersinclude, but are not limited to, e.g., polypeptides encoded by the CALCAgene, including calcitonin, procalcitonin, or calcitonin gene-relatedpeptide (CGRP). Other biomarkers associated with an IL-33-mediateddisease or disorder include, but are not limited to resistin-like alpha(RETNA); chemokine (C-C motif) ligand 8 (Ccl8); serum amyloid A 3(Saa3); Gm 1975 (BC117090); killer cell lectin-like receptor (Kirg1);stefin A1 (Csta); membrane-spanning 4-domain (Ms4a8a); chemokine (C-Cmotif) ligand 11 (Ccl11); and serine (or cysteine) peptides (Serpina3f).

Methods for detecting and/or quantifying such biomarkers are known inthe art; kits for measuring such biomarkers are available from variouscommercial sources; and various commercial diagnostic laboratories offerservices, which provide measurements of such biomarkers as well.

In one embodiment, the biomarker is calcitonin and the level ofcalcitonin is increased in a subject suffering from, or prone todeveloping an IL-33 mediated disease or disorder. In one embodiment, thebiomarker is procalcitonin and the level of procalcitonin is increasedin a subject suffering from, or prone to developing an IL-33 mediateddisease or disorder. In a related embodiment, the biomarker iscalcitonin gene-related peptide (CGRP) and the level of CGRP isincreased in a subject suffering from, or prone to developing an IL-33mediated disease or disorder. The level of calcitonin, procalcitonin, orCGRP may be assessed using any method known to those skilled in the art.Methods of detection include an immunoassay using any label fordetection, such as a radioisotopic label, an enzymatic label, or achemiluminescent label. The interpretation of the results must take intoaccount the method used, the subject's age, gender and weight (Seed'Herbomez, M. et. al., (2007), Eur. J. Endocrinology 157:749-755).Generally, a “normal” reference value of calcitonin in males is lessthan about 16 pg/mL and less than about 8 pg/mL for females. Morerecently, Camacho et. al. developed a new immunofluorometric assay forserum calcitonin and have validated it in samples from 794 patients. Theresults obtained using this assay showed that the normal cut-off rangefor males was less than 11.1 pg/mL and less than 5.5 pg/mL for females.(See Camacho, et. al., (2014), Eur. Thyroid J., Jun;3(2):117-24. A“reference” range of procalcitonin in adults and children older than 72hours is about 0.15 ng/mL or less. In healthy adults, the referencerange of procalcitonin is below the level of detection (See Dandona, P.et. al. (1994), J. Clin. Endocrinol. Metab. Dec. 79(6):1605-8.). Anormal range of CGRP in healthy individuals is generally below about 45pg/ml. As such, a value above that may be considered indicative of thepresence of a disease or disorder.

An “IL-33 associated disease parameter” may include, but not be limitedto, an increase in infiltration of neutrophils, eosinophils or ST2+ CD4T cells to a tissue (e.g. lung), goblet cells metaplasia, an increase inST2 levels in the tissue or an increase in the level of a cytokine, suchas IL-1β, IL-4, IL-5, IL-6, IL-9, IL-13, IL-33, MCP-1, TNFα in thetissue.

According to certain aspects of the invention, methods for treating anIL-33-mediated disease or disorder are provided which comprise: (a)selecting a subject who exhibits a level of at least one biomarker priorto or at the time of treatment which signifies the disease state, and(b) administering to the subject a pharmaceutical composition comprisinga therapeutically effective amount of an IL-33 antagonist. In oneembodiment of this aspect of the invention, the subject is selected onthe basis of an elevated level of calcitonin. In one embodiment, theIL-33 antagonist is an isolated monoclonal antibody the specificallybinds to IL-33, or a receptor based IL-33 antagonist (an IL-33 trap asdescribed herein).

According to other aspects of the invention, methods for treating anIL-33-mediated disease or disorder are provided which compriseadministering to a subject a pharmaceutical composition comprising atherapeutically effective amount of an IL-33 antagonist, whereinadministration of the pharmaceutical composition to the subject resultsin a decrease in at least one biomarker (e.g., calcitonin (Calca),procalcitonin, calcitonin gene-related peptide (CGRP), resistin-likealpha (RETNA), chemokine (C-C motif) ligand 8 (Ccl8), serum amyloid A 3(Saa3), Gm 1975 (BC117090), killer cell lectin-like receptor (Kirg1),stefin A1 (Csta), membrane-spanning 4-domain (Ms4a8a), chemokine (C-Cmotif) ligand 11 (Ccl11), and serine (or cysteine) peptides (Serpina3f),etc.) at a time after administration of the pharmaceutical composition,as compared to the level of the biomarker in the subject prior to theadministration.

As will be appreciated by a person of ordinary skill in the art, anincrease or decrease in a biomarker associated with an IL-33 mediateddisease or disorder can be determined by comparing (i) the level of thebiomarker measured in a subject at a defined time point afteradministration of the pharmaceutical composition comprising an IL-33antagonist to (ii) the level of the biomarker measured in the patientprior to the administration of the pharmaceutical composition comprisingan IL-33 antagonist (i.e., the “baseline measurement”). The defined timepoint at which the biomarker is measured can be, e.g., at about 4 hours,8 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 8 days, 9 days, 10 days, 14 days, 15 days, 20 days, 35 days, 40days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 84days, 85 days, or more after administration of the of the pharmaceuticalcomposition comprising an IL-33 antagonist.

According to certain particular embodiments of the present invention, asubject may exhibit a decrease in the level of one or more ofcalcitonin, procalcitonin, CGRP, resistin-like alpha (RETNA), chemokine(C-C motif) ligand 8 (Ccl8), serum amyloid A 3 (Saa3), Gm1975(BC117090), killer cell lectin-like receptor (Kirg1), stefin A1 (Csta),membrane-spanning 4-domain (Ms4a8a), chemokine (C-C motif) ligand 11(Ccl11), and serine (or cysteine) peptides (Serpina3f) followingadministration of a pharmaceutical composition comprising an IL-33antagonist (e.g., an anti-IL-33 antibody, or an IL-33 receptor basedantagonist (an IL-33 trap). For example, at about day 4, day 8, day 14,day 15, day 22, day 25, day 29, day 36, day 43, day 50, day 57, day 64,day 71, day 84, or day 85, following administration of a first, second,third or fourth dose of a pharmaceutical composition comprising about ananti-hIL-33 antagonist. The dose of an anti-IL-33 antagonistadministered to a patient may vary depending upon the age and the sizeof the patient, target disease, conditions, route of administration, andthe like. The preferred dose is typically calculated according to bodyweight or body surface area. When an IL-33 antagonist is used fortreating a condition or disease associated with IL-33 activity in anadult patient, it may be advantageous to intravenously administer theanti-IL-33 antagonist normally at a dose of about 0.01 to about 20 mg/kgbody weight, more preferably about 0.02 to about 7, about 0.03 to about5, or about 0.05 to about 3 mg/kg body weight.

The subject, according to the present invention, may exhibit a decreasein at least one of the following biomarkers: a polypeptide encoded bythe CALCA gene, selected from the group consisting of calcitonin,procalcitonin and CGRP, or may exhibit a decrease in at least one of thefollowing biomarkers: resistin-like alpha (RETNA); chemokine (C-C motif)ligand 8 (Ccl8); serum amyloid A 3 (Saa3); Gm 1975 (BC117090); killercell lectin-like receptor (Kirg1); stefin A1 (Csta); membrane-spanning4-domain (Ms4a8a); chemokine (C-C motif) ligand 11 (Ccl11); and serine(or cysteine) peptides (Serpina3f), of about 1%, 2%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95% or more from baseline (wherein “baseline” is defined as the level ofbiomarker in the subject just prior to the first administration).Similarly, at about day 4, day 8, day 14, day 15, day 22, day 25, day29, day 36, day 43, day 50, day 57, day 64, day 71, day 84 or day 85,following administration of a first, second, third or fourth dose of apharmaceutical composition of an anti-hIL-33 antagonist, the subject,according to the present invention, may exhibit a decrease in at leastone of the biomarkers described above of about 1%, 2%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95% or more from baseline (wherein “baseline” is defined as thelevel of biomarker in the subject just prior to the firstadministration).

The present invention also includes methods for determining whether asubject is a suitable subject (e.g., a subject who is likely to respondfavorably to therapy with an IL-33 antagonist) for whom administrationof a pharmaceutical composition comprising an IL-33 antagonist would bebeneficial. For example, if an individual, prior to receiving apharmaceutical composition comprising an IL-33 antagonist, exhibits alevel of a biomarker associated with an IL-33-mediated disease ordisorder, which signifies the disease state, the individual is thereforeidentified as a suitable patient for whom administration of apharmaceutical composition of the invention (a composition comprising ananti-IL-33 antagonist) would be beneficial. According to certainexemplary embodiments, an individual may be identified as a goodcandidate for anti-IL-33 therapy if the individual exhibits one or moreof the following: (i) a calcitonin level greater than about 5 pg/mL,greater than about 10 pg/mL, greater than about 20 pg/mL, greater thanabout 30 pg/mL, greater than about 40 pg/mL, greater than about 50pg/mL, greater than about 60 pg/mL, greater than about 70 pg/mL, greaterthan about 80 pg/mL, greater than about 90 pg/mL, greater than about 100pg/mL, or greater than about 200 pg/mL. Additional criteria, such asother clinical indicators of an IL-33-mediated disease or disorder maybe used in combination with any of the biomarkers described herein toidentify an individual as a suitable candidate for anti-IL-33 therapy asdescribed elsewhere herein (e.g., increase in infiltration ofneutrophils, eosinophils or ST2+ CD4 T cells to a tissue (e.g. lung),goblet cells metaplasia, an increase in ST2 levels in the tissue or anincrease in the level of a cytokine, such as IL-1β, IL-4, IL-5, IL-6,IL-9, IL-13, IL-33, MCP-1, TNFα in the tissue, as well as interferongamma).

In other aspects of the invention, biomarker levels and/or changes inbiomarker levels with treatment may have predictive value for efficacyof anti-IL33 therapy with particular anti-IL33 agents.

An additional aspect of the invention provides methods of determiningwhether a subject is likely to have, or likely to develop an IL-33mediated disease or disorder, wherein the method comprises obtaining abiological sample from the subject and measuring the level of at leastone of the biomarkers described herein; wherein the elevated level of atleast one biomarker as compared to the level of the biomarker from asubject not having an IL-33 mediated disease or disorder, or not likelyto develop an IL-33 mediated disease or disorder, identifies the subjectas a subject who is likely to have, or develop an IL-33 mediated diseaseor disorder.

An additional aspect of the invention features methods of treating anIL-33-mediated disease or disorder in a subject comprisingadministration of an anti-IL33 antagonist, wherein the subject has beendiagnosed with an IL-33-mediated disease or disorder, has been treatedwith the anti-IL33 antagonist, and has been selected for furthertreatment with the IL-33 antagonist on the basis of exhibiting reducedexpression of a biomarker (e.g. calcitonin), wherein the reducedexpression of the biomarker is determined based on a comparison to thelevel of expression of the respective biomarker in the subject prior totreatment with the anti-IL-33 antagonist. A particular aspect of theinvention features methods of treating an IL-33-mediated disease ordisorder in a subject comprising administration of an anti-IL33antagonist, wherein the subject has been diagnosed with anIL-33-mediated disease or disorder, has been treated with the anti-IL33antagonist, and has been selected for further treatment with the IL-33antagonist on the basis of exhibiting reduced expression of thebiomarker, wherein the reduced expression of the biomarker in comparisonto pre-treatment expression levels is equal to or greater than about a10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% reduction in biomarkerexpression.

Interleukin-33 Antagonists

As disclosed in detail above, the present invention includes methods,which comprise administering to a subject in need thereof a therapeuticcomposition comprising an IL-33 antagonist. As used herein, an “IL-33antagonist” is any agent, which binds to or interacts with IL-33, orwith its receptor and inhibits the normal biological signaling functionof IL-33 when IL-33 is expressed on a cell in vitro or in vivo.Non-limiting examples of categories of IL-33 antagonists include smallmolecule IL-33 antagonists, anti-IL-33 aptamers, peptide-based IL-33antagonists (e.g., “peptibody” molecules), antibodies or antigen-bindingfragments of antibodies that specifically bind human IL-33, or receptorbased IL-33 antagonists (e.g. an IL-3 trap), such as those describedherein.

In one embodiment, the present invention includes methods that compriseadministering to a patient a human antibody, or an antigen-bindingfragment thereof, that binds specifically to hIL-33. As used herein, theterm “hIL-33” means a human cytokine that specifically binds to itsreceptor, ST2. The amino acid sequence of human ST2 is shown in SEQ IDNO: 334. The amino acid sequence of human IL-33 is shown in SEQ ID NO:306. The nucleic acid encoding human IL-33 is shown in SEQ ID NO: 305.The anti-IL-33 antibodies may be selected from any one or more of thosehaving the amino acid sequences described in Table 1.

In one embodiment, the present invention includes methods that compriseadministering to a patient a receptor based IL-33 antagonist, forexample, an IL-33 trap, such as those described herein. Five differentexemplary IL-33 traps of the invention were constructed using standardmolecular biological techniques. Table 2a sets forth a summarydescription of the different IL-33 traps and their component parts.Table 2b sets forth the amino acid sequences of the IL-33 traps andtheir component parts.

The term “antibody,” as used herein, is intended to refer toimmunoglobulin molecules comprising four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds,as well as multimers thereof (e.g., IgM). Each heavy chain comprises aheavy chain variable region (abbreviated herein as HCVR or V_(H)) and aheavy chain constant region. The heavy chain constant region comprisesthree domains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises alight chain variable region (abbreviated herein as LCVR or V_(L)) and alight chain constant region. The light chain constant region comprisesone domain (C_(L)1). The V_(H) and V_(L) regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDRs), interspersed with regions that are moreconserved, termed framework regions (FR). Each V_(H) and V_(L) iscomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. In different embodiments of the invention, the FRs of theanti-IL-33 antibody (or antigen-binding portion thereof) may beidentical to the human germline sequences, or may be naturally orartificially modified. An amino acid consensus sequence may be definedbased on a side-by-side analysis of two or more CDRs.

The term “antibody,” as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDR,which is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

The constant region of an antibody is important in the ability of anantibody to fix complement and mediate cell-dependent cytotoxicity.Thus, the isotype of an antibody may be selected on the basis of whetherit is desirable for the antibody to mediate cytotoxicity.

The term “human antibody”, as used herein, is intended to includenon-naturally occurring human antibodies. The term includes antibodiesthat are recombinantly produced in a non-human mammal, or in cells of anon-human mammal. The term is not intended to include antibodiesisolated from or generated in a human subject.

The term “recombinant human antibody,” as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther below), antibodies isolated from a recombinant, combinatorialhuman antibody library (described further below), antibodies isolatedfrom an animal (e.g., a mouse) that is transgenic for humanimmunoglobulin genes (see e.g., Taylor et. al. (1992) Nucl. Acids Res.20:6287-6295) or antibodies prepared, expressed, created or isolated byany other means that involves splicing of human immunoglobulin genesequences to other DNA sequences. Such recombinant human antibodies havevariable and constant regions derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies are subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

Human antibodies can exist in two forms that are associated with hingeheterogeneity. In one form, an immunoglobulin molecule comprises astable four chain construct of approximately 150-160 kDa in which thedimers are held together by an interchain heavy chain disulfide bond. Ina second form, the dimers are not linked via inter-chain disulfide bondsand a molecule of about 75-80 kDa is formed composed of a covalentlycoupled light and heavy chain (half-antibody). These forms have beenextremely difficult to separate, even after affinity purification.

The frequency of appearance of the second form in various intact IgGisotypes is due to, but not limited to, structural differencesassociated with the hinge region isotype of the antibody. A single aminoacid substitution in the hinge region of the human IgG4 hinge cansignificantly reduce the appearance of the second form (Angal et. al.(1993) Molecular Immunology 30:105) to levels typically observed using ahuman IgG1 hinge. The instant invention encompasses antibodies havingone or more mutations in the hinge, C_(H)2 or C_(H)3 region which may bedesirable, for example, in production, to improve the yield of thedesired antibody form.

An “isolated antibody,” as used herein, means an antibody that has beenidentified and separated and/or recovered from at least one component ofits natural environment. For example, an antibody that has beenseparated or removed from at least one component of an organism, or froma tissue or cell in which the antibody naturally exists or is naturallyproduced, is an “isolated antibody” for purposes of the presentinvention. An isolated antibody also includes an antibody in situ withina recombinant cell. Isolated antibodies are antibodies that have beensubjected to at least one purification or isolation step. According tocertain embodiments, an isolated antibody may be substantially free ofother cellular material and/or chemicals.

The term “specifically binds,” or the like, means that an antibody orantigen-binding fragment thereof forms a complex with an antigen that isrelatively stable under physiologic conditions. Methods for determiningwhether an antibody specifically binds to an antigen are well known inthe art and include, for example, equilibrium dialysis, surface plasmonresonance, and the like. For example, an antibody that “specificallybinds” IL-33, as used in the context of the present invention, includesantibodies that bind IL-33 or portion thereof with a K_(D) of less thanabout 1000 nM, less than about 500 nM, less than about 300 nM, less thanabout 200 nM, less than about 100 nM, less than about 90 nM, less thanabout 80 nM, less than about 70 nM, less than about 60 nM, less thanabout 50 nM, less than about 40 nM, less than about 30 nM, less thanabout 20 nM, less than about 10 nM, less than about 5 nM, less thanabout 4 nM, less than about 3 nM, less than about 2 nM, less than about1 nM or less than about 0.5 nM, as measured in a surface plasmonresonance assay. An isolated antibody that specifically binds humanIL-33 may, however, have cross-reactivity to other antigens, such asIL-33 molecules from other (non-human) species.

The anti-IL-33 antibodies useful for the methods of the presentinvention may comprise one or more amino acid substitutions, insertionsand/or deletions in the framework and/or CDR regions of the heavy andlight chain variable domains as compared to the corresponding germlinesequences from which the antibodies were derived. Such mutations can bereadily ascertained by comparing the amino acid sequences disclosedherein to germline sequences available from, for example, publicantibody sequence databases. The present invention includes methodsinvolving the use of antibodies, and antigen-binding fragments thereof,which are derived from any of the amino acid sequences disclosed herein,wherein one or more amino acids within one or more framework and/or CDRregions are mutated to the corresponding residue(s) of the germlinesequence from which the antibody was derived, or to the correspondingresidue(s) of another human germline sequence, or to a conservativeamino acid substitution of the corresponding germline residue(s) (suchsequence changes are referred to herein collectively as “germlinemutations”). A person of ordinary skill in the art, starting with theheavy and light chain variable region sequences disclosed herein, caneasily produce numerous antibodies and antigen-binding fragments whichcomprise one or more individual germline mutations or combinationsthereof. In certain embodiments, all of the framework and/or CDRresidues within the V_(H) and/or V_(L) domains are mutated back to theresidues found in the original germline sequence from which the antibodywas derived. In other embodiments, only certain residues are mutatedback to the original germline sequence, e.g., only the mutated residuesfound within the first 8 amino acids of FR1 or within the last 8 aminoacids of FR4, or only the mutated residues found within CDR1, CDR2 orCDR3. In other embodiments, one or more of the framework and/or CDRresidue(s) are mutated to the corresponding residue(s) of a differentgermline sequence (i.e., a germline sequence that is different from thegermline sequence from which the antibody was originally derived).Furthermore, the antibodies of the present invention may contain anycombination of two or more germline mutations within the frameworkand/or CDR regions, e.g., wherein certain individual residues aremutated to the corresponding residue of a particular germline sequencewhile certain other residues that differ from the original germlinesequence are maintained or are mutated to the corresponding residue of adifferent germline sequence. Once obtained, antibodies andantigen-binding fragments that contain one or more germline mutationscan be easily tested for one or more desired property such as, improvedbinding specificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogenicity, etc. The use of antibodies and antigen-bindingfragments obtained in this general manner are encompassed within thepresent invention.

The present invention also includes methods involving the use ofanti-IL-33 antibodies comprising variants of any of the HCVR, LCVR,and/or CDR amino acid sequences disclosed herein having one or moreconservative substitutions. For example, the present invention includesthe use of anti-IL-33 antibodies having HCVR, LCVR, and/or CDR aminoacid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 orfewer, etc. conservative amino acid substitutions relative to any of theHCVR, LCVR, and/or CDR amino acid sequences disclosed herein.

The term “surface plasmon resonance,” as used herein, refers to anoptical phenomenon that allows for the analysis of real-timeinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore™ system(Biacore Life Sciences division of GE Healthcare, Piscataway, N.J.).

The term “K_(D),” as used herein, is intended to refer to theequilibrium dissociation constant of a particular antibody-antigeninteraction.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstance, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

Preparation of Human Antibodies

Methods for generating human antibodies in transgenic mice are known inthe art. Any such known methods can be used in the context of thepresent invention to make human antibodies that specifically bind tohuman IL-33.

Using VELOCIMMUNE™ technology (see, for example, U.S. Pat. No.6,596,541, Regeneron Pharmaceuticals) or any other known method forgenerating monoclonal antibodies, high affinity chimeric antibodies toIL-33 are initially isolated having a human variable region and a mouseconstant region. The VELOCIMMUNE® technology involves generation of atransgenic mouse having a genome comprising human heavy and light chainvariable regions operably linked to endogenous mouse constant regionloci such that the mouse produces an antibody comprising a humanvariable region and a mouse constant region in response to antigenicstimulation. The DNA encoding the variable regions of the heavy andlight chains of the antibody are isolated and operably linked to DNAencoding the human heavy and light chain constant regions. The DNA isthen expressed in a cell capable of expressing the fully human antibody.

Generally, a VELOCIMMUNE® mouse is challenged with the antigen ofinterest, and lymphatic cells (such as B-cells) are recovered from themice that express antibodies. The lymphatic cells may be fused with amyeloma cell line to prepare immortal hybridoma cell lines, and suchhybridoma cell lines are screened and selected to identify hybridomacell lines that produce antibodies specific to the antigen of interest.DNA encoding the variable regions of the heavy chain and light chain maybe isolated and linked to desirable isotypic constant regions of theheavy chain and light chain. Such an antibody protein may be produced ina cell, such as a CHO cell. Alternatively, DNA encoding theantigen-specific chimeric antibodies or the variable domains of thelight and heavy chains may be isolated directly from antigen-specificlymphocytes.

Initially, high affinity chimeric antibodies are isolated having a humanvariable region and a mouse constant region. The antibodies arecharacterized and selected for desirable characteristics, includingaffinity, selectivity, epitope, etc., using standard procedures known tothose skilled in the art. The mouse constant regions are replaced with adesired human constant region to generate the fully human antibody ofthe invention, for example wild-type or modified IgG1 or IgG4. While theconstant region selected may vary according to specific use, highaffinity antigen-binding and target specificity characteristics residein the variable region.

In general, the antibodies that can be used in the methods of thepresent invention possess high affinities, as described above, whenmeasured by binding to antigen either immobilized on solid phase or insolution phase. The mouse constant regions are replaced with desiredhuman constant regions to generate the fully human antibodies of theinvention. While the constant region selected may vary according tospecific use, high affinity antigen-binding and target specificitycharacteristics reside in the variable region.

Specific examples of human antibodies or antigen-binding fragments ofantibodies that specifically bind IL-33 which can be used in the contextof the methods of the present invention include any antibody orantigen-binding fragment which comprises the three heavy chain CDRs(HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region(HCVR) having an amino acid sequence selected from the group consistingof SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194,210, 226, 242, 258, 274, 290, and 308. The antibody or antigen-bindingfragment may comprise the three light chain CDRs (LCVR1, LCVR2, LCVR3)contained within a light chain variable region (LCVR) having an aminoacid sequence selected from the group consisting of SEQ ID NOs: 10, 26,42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266,282, 298, and 316. Methods and techniques for identifying CDRs withinHCVR and LCVR amino acid sequences are well known in the art and can beused to identify CDRs within the specified HCVR and/or LCVR amino acidsequences disclosed herein. Exemplary conventions that can be used toidentify the boundaries of CDRs include, e.g., the Kabat definition, theChothia definition, and the AbM definition. In general terms, the Kabatdefinition is based on sequence variability, the Chothia definition isbased on the location of the structural loop regions, and the AbMdefinition is a compromise between the Kabat and Chothia approaches.See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,”National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et.al., J. Mol. Biol. 273:927-948 (1997); and Martin et. al., Proc. Natl.Acad. Sci. USA 86:9268-9272 (1989). Public databases are also availablefor identifying CDR sequences within an antibody.

In certain embodiments of the present invention, the antibody orantigen-binding fragment thereof comprises the six CDRs (HCDR1, HCDR2,HCDR3, LCDR1, LCDR2 and LCDR3) from the heavy and light chain variableregion amino acid sequence pairs (HCVR/LCVR) selected from the groupconsisting of SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90,98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/218,226/234, 242/250, 258/266, 274/282, 290/298, and 308/316.

In certain embodiments of the present invention, the antibody orantigen-binding fragment thereof comprises HCVR/LCVR amino acid sequencepairs selected from the group consisting of SEQ ID NOs: 2/10, 18/26,34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154, 162/170,178/186, 194/202, 210/218, 226/234, 242/250, 258/266, 274/282, 290/298,and 308/316.

The study summarized in the attached Examples utilized an anti-h IL-33antibody referred to as H4H9675P. This antibody comprises an HCVR/LCVRamino acid sequence pair having SEQ ID NOs: 274/282, andHCDR1-HCDR2-HCDR3/LCDR1-LCDR2-LCDR3 domains represented by SEQ ID NOs:276-278-280/SEQ ID NOs: 284-286-288. However, the methods of the presentinvention can be practiced using any anti-IL-33 antibody disclosedherein, as well as variants and antigen-binding fragments of suchantibody.

Pharmaceutical Compositions

The present invention includes methods, which comprise administering anIL-33 antagonist to a patient, wherein the IL-33 antagonist is containedwithin a pharmaceutical composition. The pharmaceutical compositions ofthe invention are formulated with suitable carriers, excipients, andother agents that provide suitable transfer, delivery, tolerance, andthe like. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. See also Powell et. al.“Compendium of excipients for parenteral formulations” PDA (1998) JPharm Sci Technol 52:238-311.

The dose of antibody administered to a patient according to the methodsof the present invention may vary depending upon the age and the size ofthe patient, symptoms, conditions, route of administration, and thelike. The dose is typically calculated according to body weight or bodysurface area. Depending on the severity of the condition, the frequencyand the duration of the treatment can be adjusted. Effective dosages andschedules for administering pharmaceutical compositions comprisinganti-IL-33 antibodies may be determined empirically; for example,patient progress can be monitored by periodic assessment, and the doseadjusted accordingly. Moreover, interspecies scaling of dosages can beperformed using well-known methods in the art (e.g., Mordenti et. al.,1991, Pharmaceut. Res. 8:1351). Specific exemplary doses of anti-IL33antibodies, and administration regimens involving the same, that can beused in the context of the present invention are disclosed elsewhereherein.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et. al., 1987, J. Biol. Chem. 262:4429-4432). Methods ofadministration include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The composition may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but are notlimited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis,Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark),NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (BectonDickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPENSTARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to nameonly a few. Examples of disposable pen delivery devices havingapplications in subcutaneous delivery of a pharmaceutical composition ofthe present invention include, but are not limited to the SOLOSTAR™ pen(sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK^(™) Autoinjector (Amgen, Thousand Oaks, Calif.),the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.),and the HUMIRA™ Pen (Abbott Labs, Abbott Park Ill.), to name only a few.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), 1974, CRCPres., Boca Raton, Florida. In yet another embodiment, a controlledrelease system can be placed in proximity of the composition's target,thus requiring only a fraction of the systemic dose (see, e.g., Goodson,1984, in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138). Other controlled release systems are discussed in the reviewby Langer, 1990, Science 249:1527-1533.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by knownmethods. For example, the injectable preparations may be prepared, e.g.,by dissolving, suspending or emulsifying the antibody or its saltdescribed above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared can be filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc.

Exemplary pharmaceutical compositions comprising an anti-IL-33 antibodythat can be used in the context of the present invention are disclosed,e.g., in US Patent Application Publication No. 2014/0271658.

Dosage

The amount of IL-33 antagonist (e.g., anti-IL-33 antibody) administeredto a subject according to the methods of the present invention is,generally, a therapeutically effective amount. As used herein, thephrase “therapeutically effective amount” means an amount of IL-33antagonist that results in a detectable improvement in one or moresymptoms or indicia of an IL-33 mediated disease or disorder. A“therapeutically effective amount” also includes an amount of IL-33antagonist that inhibits, prevents, lessens, or delays the progressionof such disease or disorder in a subject.

In the case of an anti-IL-33 antibody, a therapeutically effectiveamount can be from about 0.05 mg to about 600 mg, e.g., about 0.05 mg,about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 10 mg,about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570mg, about 580 mg, about 590 mg, or about 600 mg, of the anti-IL-33antibody. In certain embodiments, 75 mg, 150 mg, 200 mg, or 300 mg of ananti-IL-33 antibody is administered to a subject.

The amount of IL-33 antagonist contained within the individual doses maybe expressed in terms of milligrams of antibody per kilogram of patientbody weight (i.e., mg/kg). For example, the IL-33 antagonist may beadministered to a patient at a dose of about 0.0001 to about 10 mg/kg ofpatient body weight.

Combination Therapies

The methods of the present invention, according to certain embodiments,comprise administering to the subject one or more additional therapeuticagents in combination with the IL-33 antagonist. As used herein, theexpression “in combination with” means that the additional therapeuticagents are administered before, after, or concurrent with thepharmaceutical composition comprising the IL-33 antagonist.

For example, when administered “before” the pharmaceutical compositioncomprising the IL-33 antagonist, the additional therapeutic agent may beadministered about 72 hours, about 60 hours, about 48 hours, about 36hours, about 24 hours, about 12 hours, about 10 hours, about 8 hours,about 6 hours, about 4 hours, about 2 hours, about 1 hour, about 30minutes, about 15 minutes or about 10 minutes prior to theadministration of the pharmaceutical composition comprising the IL-33antagonist. When administered “after” the pharmaceutical compositioncomprising the IL-33 antagonist, the additional therapeutic agent may beadministered about 10 minutes, about 15 minutes, about 30 minutes, about1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours,about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48hours, about 60 hours or about 72 hours after the administration of thepharmaceutical composition comprising the IL-33 antagonist.

Administration “concurrent” with the pharmaceutical compositioncomprising the IL-33 antagonist means that the additional therapeuticagent is administered to the subject in a separate dosage form withinless than 5 minutes (before, after, or at the same time) ofAdministration of the pharmaceutical composition comprising the IL-33antagonist, or administered to the subject as a single combined dosageformulation comprising both the additional therapeutic agent and theIL-33 antagonist.

The present invention includes methods of treating an IL-33 mediateddisease or disorder, which comprise administering to a patient in needof such treatment an anti-hIL-33 antibody, or an IL-33 trap incombination with at least one additional therapeutic agent. Examples ofadditional therapeutic agents which can be administered in combinationwith an anti-hIL-33 antibody in the practice of the methods of thepresent invention include, but are not limited to a non-steroidalanti-inflammatory (NSAID), a steroid, a corticosteroid (inhaled ortopical), an immunosuppressant (e.g. cyclophosphamide), ananticholinergic agent (e.g. tiotropium), a muscarinic agent (e.g.glycopyrronium), a phosphodiesterase inhibitor (e.g. theophylline,roflumilast, cilomilast), a beta blocker, cyclosporine, tacrolimus,pimecrolimus, azathioprine, methotrexate, cromolyn sodium, a proteinaseinhibitor, a bronchial dilator, a beta-2-agonist, an antihistamine,epinephrine, a decongestant, a leukotriene inhibitor, a mast cellinhibitor, a thymic stromal lymphopoietin (TSLP) antagonist, a TNFantagonist, an IgE antagonist, an IL-1 antagonist, an IL-4 or IL-4Rantagonist, an IL-13 or IL-13R antagonist, an IL-4/IL-13 dualantagonist, an IL-5 antagonist, an IL-6 or IL-6R antagonist, anantagonist of IL-8, an IL-9 antagonist, an IL-12/23 antagonist, an IL-22antagonist, an IL-17 antagonist, an IL-31 antagonist, an oral PDE4inhibitor, an IL-25 antagonist and another IL-33 antagonist or adifferent antibody or receptor based antagonist to IL-33, and any othercompound known to treat, prevent, or ameliorate an IL-33 mediateddisease or disorder in a human subject. For example, for concurrentadministration, a pharmaceutical formulation can be made which containsboth an anti-hIL-33 antibody and at least one additional therapeuticagent. The amount of the additional therapeutic agent that isadministered in combination with the anti-hIL-33 antibody in thepractice of the methods of the present invention can be easilydetermined using routine methods known and readily available in the art.

Administration Regimens

The present invention includes methods comprising administering to asubject a pharmaceutical composition comprising an IL-33 antagonist at adosing frequency of about four times a week, twice a week, once a week,once every two weeks, once every three weeks, once every four weeks,once every five weeks, once every six weeks, once every eight weeks,once every twelve weeks, or less frequently so long as a therapeuticresponse is achieved. In certain embodiments involving theadministration of a pharmaceutical composition comprising an anti-IL-33antibody, once a week dosing at an amount of about 0.01 to about 20mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 toabout 5, or about 0.05 to about 3 mg/kg body weight.

According to certain embodiments of the present invention, multipledoses of an IL-33 antagonist may be administered to a subject over adefined time course. The methods according to this aspect of theinvention comprise sequentially administering to a subject multipledoses of an IL-33 antagonist. As used herein, “sequentiallyadministering” means that each dose of IL-33 antagonist is administeredto the subject at a different point in time, e.g., on different daysseparated by a predetermined interval (e.g., hours, days, weeks ormonths). The present invention includes methods which comprisesequentially administering to the patient a single initial dose of anIL-33 antagonist, followed by one or more secondary doses of the IL-33antagonist, and optionally followed by one or more tertiary doses of theIL-33 antagonist.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the IL-33 antagonist.Thus, the “initial dose” is the dose which is administered at thebeginning of the treatment regimen (also referred to as the “baselinedose”); the “secondary doses” are the doses which are administered afterthe initial dose; and the “tertiary doses” are the doses which areadministered after the secondary doses. The initial, secondary, andtertiary doses may all contain the same amount of IL-33 antagonist, butgenerally may differ from one another in terms of frequency ofadministration. In certain embodiments, however, the amount of IL-33antagonist contained in the initial, secondary and/or tertiary dosesvaries from one another (e.g., adjusted up or down as appropriate)during the course of treatment. In certain embodiments, two or more(e.g., 2, 3, 4, or 5) doses area at the beginning of the treatmentregimen as “loading doses” followed by subsequent doses that areadministered on a less frequent basis (e.g., “maintenance doses”).

In one exemplary embodiment of the present invention, each secondaryand/or tertiary dose is administered 1 to 14 (e.g., 1, 1½, 2, 2½, 3, 3½,4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13,13½, 14, 14½, or more) weeks after the immediately preceding dose. Thephrase “the immediately preceding dose,” as used herein, means, in asequence of multiple administrations, the dose of IL-33 antagonist whichis administered to a patient prior to the administration of the verynext dose in the sequence with no intervening doses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof an IL-33 antagonist. For example, in certain embodiments, only asingle secondary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondarydoses are administered to the patient. Likewise, in certain embodiments,only a single tertiary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiarydoses are administered to the patient.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to2 weeks after the immediately preceding dose. Similarly, in embodimentsinvolving multiple tertiary doses, each tertiary dose may beadministered at the same frequency as the other tertiary doses. Forexample, each tertiary dose may be administered to the patient 2 to 4weeks after the immediately preceding dose. Alternatively, the frequencyat which the secondary and/or tertiary doses are administered to apatient can vary over the course of the treatment regimen. The frequencyof administration may also be adjusted during the course of treatment bya physician depending on the needs of the individual patient followingclinical examination.

The present invention includes administration regimens comprising anup-titration option (also referred to herein as “dose modification”). Asused herein, an “up-titration option” means that, after receiving aparticular number of doses of an IL-33 inhibitor, if a patient has notachieved a specified improvement in one or more defined therapeuticparameters (e.g., at least a 20% improvement), or otherwise exhibits aclear lack of efficacy in the opinion of a physician or health careprovider, the dose of the IL-33 inhibitor is thereafter increased. Forexample, in the case of a therapeutic regimen comprising administrationof 150 mg doses of an anti-IL-33 antibody to a patient at a frequency ofonce every two weeks, if after 8, 10, 12, 14, 16 or more weeks, thepatient has not achieved at least a 20% improvement in one parameter, orif the patient exhibits a clear lack of efficacy in the opinion of aphysician or other health care provider, then the dose of anti-IL-33antibody is increased to e.g., 200 mg, 300 mg, or more, administeredonce every two weeks thereafter (e.g., starting at week 10, 12, 14, 16,18, or later).

Treatment Populations

The present invention includes methods, which comprise administering toa subject in need thereof a therapeutic composition comprising an IL-33antagonist. As used herein, the expression “a subject in need thereof”means a human or non-human animal that exhibits one or more symptoms orindicia of an IL-33 mediated disease or disorder, such as thosedescribed herein (e.g., inflammation, eosinophilia in the lungs,neutrophil infiltration into the lungs, elevated levels of certaincytokines in the lungs, such as, but not limited to IL-5, etc.) and/orwho has been diagnosed with any of the IL-33 mediated disease ordisorders described herein.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Generation of Human Antibodies to human IL-33

An immunogen comprising human IL-33 was administered directly, with anadjuvant to stimulate the immune response, to a VELOCIMMUNE® mousecomprising DNA encoding human Immunoglobulin heavy and kappa light chainvariable regions. The antibody immune response was monitored by anIL-33-specific immunoassay. When a desired immune response was achievedsplenocytes were harvested and fused with mouse myeloma cells topreserve their viability and form hybridoma cell lines. The hybridomacell lines were screened and selected to identify cell lines thatproduce IL-33-specific antibodies. Using this technique severalanti-IL-33 chimeric antibodies (i.e., antibodies possessing humanvariable domains and mouse constant domains) were obtained; exemplaryantibodies generated in this manner were designated as follows:H1M9559N, H1M9566N, H1M9568N and H1M9565N. The human variable domainsfrom the chimeric antibodies were subsequently cloned onto humanconstant domains to make fully human anti-IL-33 antibodies as describedherein.

Anti-IL-33 antibodies were also isolated directly from antigen-positiveB cells without fusion to myeloma cells, as described in US2007/0280945A1. Using this method, several fully human anti-IL-33antibodies (i.e., antibodies possessing human variable domains and humanconstant domains) were obtained; exemplary antibodies generated in thismanner were designated as follows: H4H9629P, H4H9633P, H4H6940P,H4H9659P, H4H9660P, H4H9662P, H4H9663P, H4H9664P, H4H9665P, H4H9666P,H4H9667P, H4H9670P, H4H9671 P, H4H9672P, H4H9675P, and H4H9676P.

Certain biological properties of the exemplary anti-IL-33 antibodiesgenerated in accordance with the methods of this Example are describedin detail in the Examples set forth below.

Example 2 Heavy and Light Chain Variable Region Amino Acid Sequences

Table 1 sets forth the heavy and light chain variable region amino acidsequence pairs, and CDR sequences, of selected anti-IL-33 antibodies andtheir corresponding antibody identifiers.

TABLE 1 Antibody SEQ ID NOs: Designation HCVR HCDR1 HCDR2 HCDR3 LCVRLCDR1 LCDR2 LCDR3 H1M9559N 2 4 6 8 10 12 14 16 H1M9566N 18 20 22 24 2628 30 32 H1M9568N 34 36 38 40 42 44 46 48 H4H9629P 50 52 54 56 58 60 6264 H4H9633P 66 68 70 72 74 76 78 80 H4H9640P 82 84 86 88 90 92 94 96H4H9659P 98 100 102 104 106 108 110 112 H4H9660P 114 116 118 120 122 124126 128 H4H9662P 130 132 134 136 138 140 142 144 H4H9663P 146 148 150152 154 156 158 160 H4H9664P 162 164 166 168 170 172 174 176 H4H9665P178 180 182 184 186 188 190 192 H4H9666P 194 196 198 200 202 204 206 208H4H9667P 210 212 214 216 218 220 222 224 H4H9670P 226 228 230 232 234236 238 240 H4H9671P 242 244 246 248 250 252 254 256 H4H9672P 258 260262 264 266 268 270 272 H4H9675P 274 276 278 280 282 284 286 288H4H9676P 290 292 294 296 298 300 302 304 H1M9565N 308 310 312 314 316318 320 322

Antibodies are typically referred to herein according to the followingnomenclature: Fc prefix (e.g. “H1M,” or “H4H”), followed by a numericalidentifier (e.g. “9559,” “9566,” or “9629” as shown in Table 1),followed by a “P,” or “N” suffix. Thus, according to this nomenclature,an antibody may be referred to herein as, e.g., “H1M9559N,” “H1M9566N,”“H4H9629P,” etc. The H1M and H4H prefixes on the antibody designationsused herein indicate the particular Fc region isotype of the antibody.For example, an “H1M” antibody has a mouse IgG1 Fc, whereas an “H4H”antibody has a human IgG4 Fc. As will be appreciated by a person ofordinary skill in the art, an antibody having a particular Fc isotypecan be converted to an antibody with a different Fc isotype (e.g., anantibody with a mouse IgG1 Fc can be converted to an antibody with ahuman IgG4, etc.), but in any event, the variable domains (including theCDRs)—which are indicated by the numerical identifiers shown in Table1—will remain the same, and the binding properties are expected to beidentical or substantially similar regardless of the nature of the Fcdomain.

Example 3 Construction of IL-33 Traps

Five different exemplary IL-33 antagonists (IL-33 traps) of theinvention were constructed using standard molecular biologicaltechniques. The first IL-33 antagonist (hST2-hFc, SEQ ID NO:323)consists of the soluble extracellular region of human ST2 (SEQ IDNO:327) fused at its C-terminus to the N-terminus of a human IgG1 Fcregion (SEQ ID NO:331). The second IL-33 antagonist (hST2-mFc, SEQ IDNO:324) consists of the soluble extracellular region of human ST2 (SEQID NO:327) fused at its C-terminus to the N-terminus of a mouse IgG2a Fcregion (SEQ ID NO:332). The third IL-33 antagonist (hST2-hIL RAcP-mFc,SEQ ID NO: 325) consists of an in-line fusion having human ST2 (SEQ IDNO:327) at its N-terminus, followed by the extracellular region of humanIL-1 RAcP (SEQ ID NO:329), followed by a mouse IgG2a Fc (SEQ ID NO:332)at its C-terminus. The fourth IL-33 antagonist (mST2-mIL1 RAcP-mFc, SEQID NO: 326) consists of an in-line fusion having mouse ST2 (SEQ IDNO:328) at its N-terminus, followed by the extracellular region of mouseIL-1 RAcP (SEQ ID NO:330), followed by a mouse IgG2a Fc (SEQ ID NO:332)at its C-terminus. The fifth IL-33 antagonist (hST2-hIL RAcP-hFc, SEQ IDNO:335) consists of an in line fusion having human ST2 of SEQ ID NO: 327at its N-terminus, followed by the extracellular region of human IL-1RAcP (SEQ ID NO: 329) followed by a human IgG1 Fc (SEQ ID NO: 331) atits C terminus. Table 2a sets forth a summary description of thedifferent IL-33 antagonists and their component parts. Table 2b setsforth the amino acid sequences of the IL-33 antagonists and theircomponent parts.

TABLE 2a Summary of IL-33 Antagonists (IL-33 traps) Amino Acid Sequenceof Full Antagonist IL-33 Antagonist Molecule D1 Component D2 Component MComponent hST2-hFc SEQ ID NO: 323 human ST2 Absent human IgG1 Fcextracellular (SEQ ID (SEQ ID NO: 331) NO: 327) hST2-mFc SEQ ID NO: 324human ST2 Absent mouse IgG2a extracellular Fc (SEQ ID (SEQ ID NO: 327)NO: 332) hST2-hIL1RAcP- SEQ ID NO: 325 human ST2 human IL-1RAcP mouseIgG2a mFc extracellular extracellular Fc (SEQ ID (SEQ ID NO: 329) (SEQID NO: 327) NO: 332) mST2-mIL1RAcP- SEQ ID NO: 326 mouse ST2 mouse IL-1RAcP mouse IgG2a mFc extracellular extracellular Fc (SEQ ID (SEQ ID NO:330) (SEQ ID NO: 328) NO: 332) hST2-hIL1RAcP- SEQ ID NO: 335 human ST2human IL-1RAcP human IgG1 Fc hFc extracellular extracellular (SEQ ID(SEQ ID (SEQ ID NO: 329) NO: 331) NO: 327)

TABLE 2b Amino Acid Sequences Identifier Sequence SEQ IDKFSKQSWGLENEALIVRCPRQGKPSYTVDWYYSQTNKSIPTQERNRVFASGQL NO: 323LKFLPAAVADSGIYTCIVRSPTFNRTGYANVTIYKKQSDCNVPDYLMYSTVSGSE (hST2-hFc)KNSKIYCPTIDLYNWTAPLEWFKNCQALQGSRYRAHKSFLVIDNVMTEDAGDYTCKFIHNENGANYSVTATRSFTVKDEQGFSLFPVIGAPAQNEIKEVEIGKNANLTCSACFGKGTQFLAAVLWQLNGTKITDFGEPRIQQEEGQNQSFSNGLACLDMVLRIADVKEEDLLLQYDCLALNLHGLRRHTVRLSRKNPIDHHSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ IDKFSKQSWGLENEALIVRCPRQGKPSYTVDWYYSQTNKSIPTQERNRVFASGQL NO: 324LKFLPAAVADSGIYTCIVRSPTFNRTGYANVTIYKKQSDCNVPDYLMYSTVSGSE (hST2-mFc)KNSKIYCPTIDLYNWTAPLEWFKNCQALQGSRYRAHKSFLVIDNVMTEDAGDYTCKFIHNENGANYSVTATRSFTVKDEQGFSLFPVIGAPAQNEIKEVEIGKNANLTCSACFGKGTQFLAAVLWQLNGTKITDFGEPRIQQEEGQNQSFSNGLACLDMVLRIADVKEEDLLLQYDCLALNLHGLRRHTVRLSRKNPIDHHSEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVICVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG K SEQ IDKFSKQSWGLENEALIVRCPRQGKPSYTVDWYYSQTNKSIPTQERNRVFASGQL NO: 325LKFLPAAVADSGIYTCIVRSPTFNRTGYANVTIYKKQSDCNVPDYLMYSTVSGSE (hST2-KNSKIYCPTIDLYNWTAPLEWFKNCQALQGSRYRAHKSFLVIDNVMTEDAGDYT hIL1RAcP-CKFIHNENGANYSVTATRSFTVKDEQGFSLFPVIGAPAQNEIKEVEIGKNANLTC mFc)SACFGKGTQFLAAVLWQLNGTKITDFGEPRIQQEEGQNQSFSNGLACLDMVLRIADVKEEDLLLQYDCLALNLHGLRRHTVRLSRKNPIDHHSSERCDDWGLDTMRQIQVFEDEPARIKCPLFEHFLKFNYSTAHSAGLTLIWYWTRQDRDLEEPINFRLPENRISKEKDVLWFRPTLLNDTGNYTCMLRNTTYCSKVAFPLEVVQKDSCFNSPMKLPVHKLYIEYGIQRITCPNVDGYFPSSVKPTITWYMGCYKIQNFNNVIPEGMNLSFLIALISNNGNYTCVVTYPENGRTFHLTRTLTVKVVGSPKNAVPPVIHSPNDHVVYEKEPGEELLIPCTVYFSFLMDSRNEVWWTIDGKKPDDITIDVTINESISHSRTEDETRTQILSIKKVTSEDLKRSYVCHARSAKGEVAKAAKVKQKVPAPRYTVESGEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVICVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHN HHTTKSFSRTPGKSEQ ID SKSSWGLENEALIVRCPQRGRSTYPVEWYYSDTNESIPTQKRNRIFVSRDRLKF NO: 326LPARVEDSGIYACVIRSPNLNKTGYLNVTIHKKPPSCNIPDYLMYSTVRGSDKNF (mST2-KITCPTIDLYNWTAPVQWFKNCKALQEPRFRAHRSYLFIDNVTHDDEGDYTCQF mIL1RAcP-THAENGTNYIVTATRSFTVEEKGFSMFPVITNPPYNHTMEVEIGKPASIACSACF mFc)GKGSHFLADVLWQINKTVVGNFGEARIQEEEGRNESSSNDMDCLTSVLRITGVTEKDLSLEYDCLALNLHGMIRHTIRLRRKQPIDHRSERCDDWGLDTMRQIQVFEDEPARIKCPLFEHFLKYNYSTAHSSGLTLIWYWTRQDRDLEEPINFRLPENRISKEKDVLWFRPTLLNDTGNYTCMLRNTTYCSKVAFPLEVVQKDSCFNSAMRFPVHKMYIEHGIHKITCPNVDGYFPSSVKPSVTWYKGCTEIVDFHNVLPEGMNLSFFIPLVSNNGNYTCVVTYPENGRLFHLTRTVTVKVVGSPKDALPPQIYSPNDRVVYEKEPGEELVIPCKVYFSFIMDSHNEVWWTIDGKKPDDVTVDITINESVSYSSTEDETRTQILSIKKVTPEDLRRNYVCHARNTKGEAEQAAKVKQKVIPPRYTVESGEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVICVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTK SFSRTPGK SEQ IDKFSKQSWGLENEALIVRCPRQGKPSYTVDWYYSQTNKSIPTQERNRVFASGQL NO: 327LKFLPAAVADSGIYTCIVRSPTFNRTGYANVTIYKKQSDCNVPDYLMYSTVSGSE (human ST2KNSKIYCPTIDLYNWTAPLEWFKNCQALQGSRYRAHKSFLVIDNVMTEDAGDYT extracellularCKFIHNENGANYSVTATRSFTVKDEQGFSLFPVIGAPAQNEIKEVEIGKNANLTC domain)SACFGKGTQFLAAVLWQLNGTKITDFGEPRIQQEEGQNQSFSNGLACLDMVLRIADVKEEDLLLQYDCLALNLHGLRRHTVRLSRKNPIDHHS SEQ IDSKSSWGLENEALIVRCPQRGRSTYPVEWYYSDTNESIPTQKRNRIFVSRDRLKF NO: 328LPARVEDSGIYACVIRSPNLNKTGYLNVTIHKKPPSCNIPDYLMYSTVRGSDKNF (mouse ST2KITCPTIDLYNWTAPVQWFKNCKALQEPRFRAHRSYLFIDNVTHDDEGDYTCQF extracellularTHAENGTNYIVTATRSFTVEEKGFSMFPVITNPPYNHTMEVEIGKPASIACSACF domain)GKGSHFLADVLWQINKTVVGNFGEARIQEEEGRNESSSNDMDCLTSVLRITGVTEKDLSLEYDCLALNLHGMIRHTIRLRRKQPIDHR SEQ IDSERCDDWGLDTMRQIQVFEDEPARIKCPLFEHFLKFNYSTAHSAGLTLIWYWTR NO: 329QDRDLEEPINFRLPENRISKEKDVLWFRPTLLNDTGNYTCMLRNTTYCSKVAFPL (humanEVVQKDSCFNSPMKLPVHKLYIEYGIQRITCPNVDGYFPSSVKPTITWYMGCYKI IL1RAcPQNFNNVIPEGMNLSFLIALISNNGNYTCVVTYPENGRTFHLTRTLTVKVVGSPKN extracellularAVPPVIHSPNDHVVYEKEPGEELLIPCTVYFSFLMDSRNEVWWTIDGKKPDDITI domain)DVTINESISHSRTEDETRTQILSIKKVTSEDLKRSYVCHARSAKGEVAKAAKVKQK VPAPRYTVESEQ ID SERCDDWGLDTMRQIQVFEDEPARIKCPLFEHFLKYNYSTAHSSGLTLIWYWTR NO: 330QDRDLEEPINFRLPENRISKEKDVLWFRPTLLNDTGNYTCMLRNTTYCSKVAFPL (mouseEVVQKDSCFNSAMRFPVHKMYIEHGIHKITCPNVDGYFPSSVKPSVTWYKGCTE IL1RAcPIVDFHNVLPEGMNLSFFIPLVSNNGNYTCVVTYPENGRLFHLTRTVTVKVVGSPK extracellularDALPPQIYSPNDRVVYEKEPGEELVIPCKVYFSFIMDSHNEVWWTIDGKKPDDV domain)TVDITINESVSYSSTEDETRTQILSIKKVTPEDLRRNYVCHARNTKGEAEQAAKVK QKVIPPRYTVESEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK NO: 331FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK (human IgG1ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW Fc)ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGKSEQ ID EPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDD NO: 332PDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKV (mouse IgG2aNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIY Fc)VEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHE GLHNHHTTKSFSRTPGKSEQ ID SITGISPITESLASLSTYNDQSITFALEDESYEIYVEDLKKDKKKDKVLLSYYESQH NO: 333PSSESGDGVDGKMLMVTLSPTKDFWLQANNKEHSVELHKCEKPLPDQAFFVLH (M. fascicularisNRSFNCVSFECKTDPGVFIGVKDNHLALIKVDYSENLGSENILFKLSEILEHHHHH IL-33-6His) HSEQ ID KFSKQSWGLENEALIVRCPRQGKPSYTVDWYYSQTNKSIPTQERNRVFASGQL NO: 335LKFLPAAVADSGIYTCIVRSPTFNRTGYANVTIYKKQSDCNVPDYLMYSTVSGSE (hST2-KNSKIYCPTIDLYNWTAPLEWFKNCQALQGSRYRAHKSFLVIDNVMTEDAGDYT hIL1RAcP-hFc)CKFIHNENGANYSVTATRSFTVKDEQGFSLFPVIGAPAQNEIKEVEIGKNANLTCSACFGKGTQFLAAVLWQLNGTKITDFGEPRIQQEEGQNQSFSNGLACLDMVLRIADVKEEDLLLQYDCLALNLHGLRRHTVRLSRKNPIDHHSSERCDDWGLDTMRQIQVFEDEPARIKCPLFEHFLKFNYSTAHSAGLTLIWYWTRQDRDLEEPINFRLPENRISKEKDVLWFRPTLLNDTGNYTCMLRNTTYCSKVAFPLEVVQKDSCFNSPMKLPVHKLYIEYGIQRITCPNVDGYFPSSVKPTITWYMGCYKIQNFNNVIPEGMNLSFLIALISNNGNYTCVVTYPENGRTFHLTRTLTVKVVGSPKNAVPPVIHSPNDHVVYEKEPGEELLIPCTVYFSFLMDSRNEVWWTIDGKKPDDITIDVTINESISHSRTEDETRTQILSIKKVTSEDLKRSYVCHARSAKGEVAKAAKVKQKVPAPRYTVEDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK

Example 4 Biomarkers for IL-33 Activity

Studies were done to determine which genes were elevated in mice thatoverexpressed IL-33. The studies described below summarize the methodsand animal models used to determine which genes correlated with IL-33expression in vivo and which genes were modulated following treatment ofthe animals with an IL-33 antagonist.

Hydrodynamic DNA delivery

Mouse IL-33 was overexpressed in wild type (WT) mice by hydrodynamic DNAdelivery (HDD). For the HDD experiment, WT mice were injected witheither 50 μg or 25 ug of a plasmid expressing full-length mouse IL-33(See GenBank accession number NM_001164724; mIL-33) or with 50 μg or 25ug of the same plasmid devoid of coding sequence (empty vector). Micewere sacrificed 7 days after the HDD injection, and blood, dorsal rootganglia (DRG), heart, kidney, liver, lung and spleen were collected. Thetop ten genes expressed in these tissues are shown in table 4 below.

Microarray analysis of collected tissue

Cy3-CTP was incorporated into amplified cRNA from 500 ng of total RNAusing QuickAmp RNA Amplification Kit (Agilent). Cy3 labeled cRNA fromeach sample was then hybridized to a custom Agilent array comprising of43538 60 mer oligos covering mouse transcriptome. The hybridization andwash of the arrays were performed according to the Agilent protocol andarrays were scanned on an Agilent Microarray scanner. The data wasextracted from scanned array images using Agilent Feature ExtractionSoftware 9.5.

Serum Collection

Whole blood was collected into Microtainer® tubes by cardiac puncture atthe end of the study. Blood was allowed to clot by leaving itundisturbed at room temperature for at least 30 minutes. Clotted bloodand cells were pelleted by centrifuging at 18,000× g for 10 minutes at4° C. in a refrigerated centrifuge. The resulting supernatant,designated serum, was transferred into clean polypropylene plates andprocessed or stored appropriately.

Determination of IL-33 Protein Concentrations in the Serum or CellLysate Extracts

ELISA kits from R&D systems were used to determine IL-33 concentrationsfor human (DY3625) and mouse (DY3626) IL-33 according to themanufacturers instructions.

Determination of Mouse Calcitonin (CT) Concentrations in the Serum orCell Lysate Extracts

Mouse calcitonin was measured in the serum of mice using a mouseCalcitonin (CT) ELISA kit from CusaBio (Through ARP) Cat#CSB-E05133m.The assay procedure was carried out according to the manufacturersinstructions provided with the kits.

Determination of Mouse IL-5 Concentrations in Cell Lysate Extracts

Cytokine concentrations in the lung protein extracts were measured usinga V-Plex custom Mouse multiplex immunoassay kit (MesoScale Discovery,#K152A41), according to the manufacturer's instructions.

House Dust Mite Induced Chronic Lung Inflammation Model

IL-33 Humin mice were intranasally administered either 50 μg house dustmite extract (HDM; Greer, #XPB70D3A2.5) diluted in 20 μL of 1× phosphatebuffered saline (PBS) or 20 μL of 1× PBS for 3 days per week for 15weeks. A second control group of IL33 Humln mice were administered 50 μgHDM extract diluted in 20 μL of 1× PBS for 3 days per week for either 4or 11 weeks, to assess the severity of the disease at the onset ofantibody treatment. Two groups of HDM challenged mice were injectedsubcutaneously with 25 mg/kg of either an anti-IL-33 antibody, H4H9675P,or an isotype control antibody starting after either 4 or 11 weeks ofHDM challenge and then twice per week until the end of the HDM challenge(8 or 4 weeks of antibody treatment). On day 85 of the study, all micewere sacrificed and their lungs were harvested. Experimental dosing andtreatment protocol for groups of mice are shown in Table 3.

IL-33 Humin mice were intranasally administered either 50 μg house dustmite extract (HDM; Greer, #XPB70D3A2.5) diluted in 20 μL of 1× phosphatebuffered saline (PBS) or 20 μL of 1× PBS for 3 days per week for 15weeks. A second control group of IL33 Humln mice were administered 50 μgHDM extract diluted in 20 μL of 1× PBS for 3 days per week for 11 weeks,to assess the severity of the disease at the onset of antibodytreatment. Two groups of HDM challenged mice were injectedsubcutaneously with 25 mg/kg of either an anti-IL-33 antibody, H4H9675P,or an isotype control antibody starting after 11 weeks of HDM challengeand then twice per week until the end of the HDM challenge (8 weeks ofantibody treatment). On day 85 of the study, all mice were sacrificedand their lungs were harvested. Experimental dosing and treatmentprotocol for groups of mice are shown in Table 3.

TABLE 3 Experimental dosing and treatment protocol for groups of miceLength of Intranasal intranasal Group Mice challenge challenge Antibody1 IL-33 Humln 1X PBS 15 weeks None mice 2 IL-33 Humln 50 μg HDM in 4 or11 weeks None mice 20 μL 1X PBS 3 IL-33 Humln 50 μg HDM in 15 weeks Nonemice 20 μL 1X PBS 4 IL-33 Humln 50 μg HDM in 15 weeks Isotype controlmice 20 μL 1X PBS antibody 5 IL-33 Humln 50 μg HDM in 15 weeksAnti-IL-33 mice 20 μL 1X PBS antibody (H4H9675P)

Tissue Harvest for Gene Expression Analysis

After exsanguination, lung was removed, placed into tubes containing 400μL of RNA Later (Ambion, Cat#AM720) and stored at −20° C. untilprocessing. Tissues were homogenized in TRIzol and chloroform was usedfor phase separation. The aqueous phase, containing total RNA, waspurified using MagMAX™-96 for Microarrays Total RNA Isolation Kitaccording to manufacturer's specifications. Genomic DNA was removedusing MagMAX™Turbo™DNase Buffer and TURBO DNase from the MagMAX kitlisted above. mRNA (up to 2.5 μg) was reverse-transcribed into cDNAusing SuperScript® VILO™ Master Mix. cDNA was diluted to 2ng/μL and 10ng cDNA was amplified with the TaqMan® Gene Expression Master Mix andthe relevant probes (mouse B2m, mouse Calca, human IL33) using the ABI7900HT Sequence Detection System (Applied Biosystems). The B2m probe wasused to amplify the beta-2 microblobulin (B2m) gene as an internalcontrol in order to normalize cDNA input differences. Data analysis wasperformed using Microsoft Excel and Graph Pad Prism™ software.Expression of each gene was normalized to B2m expression within the samesample.

Lung Harvest for Cytokine Analysis

After exsanguination, the cranial and middle lobes of the right lungfrom each mouse were removed and placed into tubes containing a solutionof tissue protein extraction reagent (1X T-PER reagent; Pierce, #78510)supplemented with 1× Halt Protease inhibitor cocktail (Pierce, #78430).All further steps were performed on ice. The volume of T-PER Reagent(containing the protease inhibitor cocktail) was adjusted for eachsample to match a 1:8 (w/v) tissue to T-PER ratio. Lung samples werehomogenized in the tubes, using the Tissue Lyser II (Qiagen, Cat#85300).The resulting lysates were centrifuged to pellet debris. Thesupernatants containing the soluble protein extracts were transferred tofresh tubes and stored at 4° C. until further analysis.

Total protein content in the lung protein extracts was measured using aBradford assay. For the assay, 10 μL of diluted extract samples wereplated into 96 well plates in duplicates and mixed with 200 μL of 1× DyeReagent (Biorad, #500-0006). Serial dilutions of bovine serum albumin(BSA; Sigma, #A7979), starting at 700 μg/mL in 1× T-Per reagent wereused as a standard to determine the protein concentration of theextracts. After a 5-minute incubation at room temperature, absorbance at595 nm was measured on a Molecular Devices SpectraMax M5 plate reader.Data analysis to determine total lung extract protein content based onthe BSA standard was performed using GraphPad Prism™ software.

Each cytokine concentration in lung total protein extracts from all micein each group was normalized to the total protein content of theextracts measured by the Bradford assay, and expressed for each group asaverage pg of cytokine per mg of total lung proteins (pg/mg lungprotein, ±SD).

TABLE 4 Top IL-33 Perturbed Genes Heart Kidney Liver Lung Spleen GeneDrgIL33 IL33 IL33 IL33 IL33 IL33 Symbol Description 10.2 415.9 33.5 72.75.0 86.3 Calca Calcitonin/calcitonin- gene related peptide (CGRP) 36.2114.8 51.7 572.8 24.9 13.8 Retnla Resistin like alpha 6.5 194.0 19.5152.2 4.5 19.4 Ccl8 Chemokine (C-C motif) ligand 8 6.5 17.8 251.0 14.98.0 45.9 Saa3 Serum amyloid A 3 38.3 28.2 8.3 66.6 53.2 29.3 BC117090Gm1975 4.9 32.8 61.2 40.4 15.3 3.6 Klrg1 Killer cell lectin-likereceptor 15.4 11.7 6.8 86.7 27.1 13.8 Csta Stefin A1 5.7 46.1 18.2 38.41.8 4.5 Ms4a8a Membrane-spanning 4-domain 1.7 19.0 23.3 81.6 9.4 6.6Ccl11 Chemokine (C-C motif) ligand 11 3.7 17.9 49.0 18.0 25.8 10.5Serpina3f Serine (or cysteine) peptides

Lung Harvest for Pulmonary Cell Infiltrate Analysis

After exsanguination, the caudal lobe of the right lung from each mousewas removed, placed into a tube containing a solution of 20 μg/mL DNaseand 0.7 U/mL Liberase TH diluted in Hank's Balanced Salt Solution (HBSS)and chopped into pieces that were approximately 2 to 3 mm in size. Thetubes with the chopped lungs were then incubated in a 37° C. water bathfor 20 minutes. The reaction was stopped by addingethylenediaminetetraacetic acid (EDTA) at a final concentration of 10mM. The samples were then transferred to gentleMACS C Tubes. The volumein the C Tubes was brought up to 3 mL with MACS buffer and the sampleswere subsequently dissociated to form single cell suspensions using agentleMACS dissociator® (Miltenyi Biotec). The tubes were thencentrifuged and the resulting pellet was resuspended in 4 mL of 1× RedBlood Cell Lysing Buffer to lyse red blood cells. After incubation for 3minutes at room temperature, 10 mL of 1 × DPBS was added to deactivatethe red blood cell lysing buffer. The cell suspensions were thencentrifuged, and the resulting cell pellets were resuspended in 1 mL of1 × DPBS. The resuspended samples were centrifuge-filtered (1 minute at400× g) through a 100 μm filter plate and approximately 1.5×10⁶ cellsper well were plated in a 96-well U-bottom plate. Cells were thencentrifuged and the cell pellets resuspended in 100 μL of Near-IRLIVE/DEAD® Fixable Dead Cell Stain (Invitrogen Cat#: L34976, Lot#:1647137) diluted at 1:500 in 1 × DPBS to determine cell viability. Cellswere incubated with the viability dye for 20 minutes at room temperaturewhile protected from light. After one wash in 1 × DPBS, cells wereincubated in 50 μL of MACS buffer containing 10 μg/mL of purified ratanti-mouse CD16/CD32 Fc Block, for 10 minutes at 4° C. The cells werethen incubated in the appropriate 2× antibody mixture diluted inBrilliant Stain Buffer (described in Table 5) for 30 minutes at 4° C.while protected from light. After antibody incubation, the cells werewashed twice in MACS buffer, resuspended in BD CytoFix that had beendiluted 1:4 in 1 × DPBS and then incubated for 15 minutes at 4° C. whileprotected from light. The cells were subsequently washed and resuspendedin MACS buffer. Cell suspensions were then filtered into a new U-Bottomplate through an AcroPrep Advance 96 Filter Plate 30-40 μm. Sample datawere acquired on a LSR Fortessa X-20 cell analyzer using the HTSattachment (BD Biosciences). Data analysis was performed using FlowJo XSoftware (Tree Star, OR) and statistical analysis was performed usingGraphPad Prism™ (GraphPad Software, CA). Eosinophils were defined aslive (cell viability dye negative), singlets, CD45⁺, F4/80⁺, Ly6G⁻,CD11c^(lo-Int), SiglecF^(hi). Neutrophils were defined as live,singlets, CD45⁺, F4/80⁻, Ly6G⁺. Data for eosinophils, and neutrophilswere expressed as frequency of live cells.

TABLE 5 Antibodies Used for Flow Cytometry Analysis Final AntibodyFluorochrome Manufacturer Catalog #, Lot # dilution CD45 Alexa FluorBioLegend 103128, B191240 1/200 700 Siglec-F BV 421 BD 562681, 42349131/200 F4/80 PE BD  56410, 5054900 1/500 Ly6G BUV395 BD 563978, 41786211/200 CD11c APC BD 550261, 5016523 1/200

Statistical Analysis

Statistical analyses were performed using Graph Pad Prism™ version 6.0(GraphPad Software, CA).

Normality of the data was evaluated using the Kolmogorov-Smirnov test.If data passed the normality test, and standard deviations of thedifferent groups were not statistically different from each other asassessed by the Brown-Forsythe test, results were interpreted by one-wayanalysis of variance (ANOVA) followed by the Tukey post hoc test formultiple comparisons. If data failed to pass the normality test, orstandard deviations were significantly different, results wereinterpreted using the Kruskal-Wallis test followed by the Dunn's posthoc test for multiple comparisons. Differences were considered to bestatistically significant when P value <0.05.

Correlation coefficient and significance were computed using two-tailedSpearman non-parametric correlation. Correlations were considered to bestatistically significant when P value <0.05.

Summary of Results:

The results demonstrated that the top ten genes that were perturbed inanimals that overexpressed IL-33 were calcitonin (Calca), resistin-likealpha (RETNA), chemokine (C-C motif) ligand 8 (Ccl8), serum amyloid A 3(Saa3), Gm 1975 (BC117090), killer cell lectin-like receptor (Kirg1),stefin A1 (Csta), membrane-spanning 4-domain (Ms4a8a), chemokine (C-Cmotif) ligand 11 (Ccl11), and serine (or cysteine) peptides (Serpina3f)(See Table 4). Moreover, the data also showed that both serum IL-33 andserum calcitonin were significantly elevated in this mouse model (SeeFIG. 1A for serum IL-33 levels and FIG. 1B for serum calcitonin levels).The increase in serum IL-33 correlated with the increase in serum levelsof calcitonin.

In addition, the chronic house dust mite (HDM) challenge model, whichwas used as a model of severe, progressive lung inflammation followingexposure to an allergen, showed that calcitonin gene expression isupregulated in the lungs of mice upon chronic HDM challenge, but theexpression of calcitonin is reduced significantly in the lungs of micethat have been treated with an IL-33 antagonist (H4H9675P anti-IL-33antibody). Treatment with an isotype matched negative control antibodyhad no effect on the level of calcitonin gene expression (FIG. 2).

In addition, serum calcitonin was also elevated in this HDM mouse modeland the level of serum calcitonin correlated with the level ofcalcitonin seen in the lungs of these mice (See FIG. 3). Moreover, thelevel of serum calcitonin correlated with the elevated levels of IL-33observed in this HDM mouse model (See FIG. 4), suggesting thatcalcitonin could be used as a potential biomarker for IL-33 in an IL-33mediated disease process (e.g. an allergic response).

In terms of IL-33 mediated disease parameters, the frequency of lungneutrophils, lung eosinophils and IL5 levels were studied in this HDMmodel. By 15 weeks after challenge with house dust mite allergen, allthree parameters were found to be elevated (See FIGS. 5A (neutrophils),5B (eosinophils) and 5C (IL-5)). However, mice that received the IL-33antibody H4H9675P showed significantly fewer neutrophils, eosinophilsand IL-5 in their lungs compared to the mice challenged with allergenand left untreated, or administered a negative isotype control antibody(See FIGS. 5A, 5B and 5C).

Furthermore, there was a significant correlation between serumcalcitonin levels and the three disease parameters studied. FIG. 6Ashows the correlation between serum calcitonin and lung neutrophils.FIG. 6B shows the correlation between serum calcitonin and lungeosinophils and FIG. 6C shows the correlation between serum calcitoninand lung IL-5 levels.

In addition, there was a significant correlation between lung IL-33levels and the frequency of lung neutrophils (FIG. 7A), lung eosinophils(FIG. 7B) and lung IL-5 levels (FIG. 7C).

In summary, the data obtained from the house dust mite model indicatethat there is a significant correlation between the level of IL-33, thefrequency of at least three disease parameters associated with thismodel of chronic allergy and the expression of calcitonin. As such, thisinformation suggests that calcitonin may be one biomarker of diseaseseverity and/or progression in a mammal and furthermore, the datasuggest that calcitonin may be used to assess the effectiveness oftherapy with an IL-33 antagonist as described herein.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

1. A method for diagnosing and treating an interleukin-33 (IL-33)mediated disease or disorder in a subject, the method comprising: (a)obtaining a biological sample from the subject and measuring a level ofa biomarker selected from calcitonin, procalcitonin or calcitoningene-related peptide (CGRP) in the sample; (b) diagnosing the subjectwith an IL-33 mediated disease or disorder when the level of calcitonin,procalcitonin or CGRP is elevated relative to a reference level of thecalcitonin, procalcitonin or CGRP, respectively; and (c) administeringto the subject a pharmaceutical composition comprising a therapeuticallyeffective amount of an interleukin-33 antagonist, wherein theinterleukin-33 antagonist is an anti-IL-33 antibody or antigen-bindingfragment thereof, or an IL-33 trap.
 2. (canceled)
 3. The method of claim1, wherein the biomarker is calcitonin.
 4. The method of claim 1,wherein the IL-33 antagonist is an anti-IL-33 antibody orantigen-binding fragment thereof.
 5. The method of claim 4, wherein theanti-IL-33 antibody or antigen-binding fragment is an isolated humanmonoclonal antibody or antigen-binding fragment thereof thatspecifically binds human interleukin-33 (IL-33), and comprises: (a) thecomplementarity determining regions (CDRs) of a heavy chain variableregion (HCVR) having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162,178, 194, 210, 226, 242, 258, 274, 290, and 308; and (b) the CDRs of alight chain variable region (LCVR) having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74,90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, and316.
 6. The method of claim 4, wherein the anti-IL-33 antibody orantigen-binding fragment thereof comprises the heavy and light chainCDRs of a HCVR/LCVR amino acid sequence pair selected from the groupconsisting of: SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90,98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 210/218,226/234, 242/250, 258/266, 274/282, 290/298, and 308/316.
 7. The methodof claim 4, wherein the antibody or antigen-binding fragment comprisesHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively, selected fromthe group consisting of: SEQ ID NOs: 4-6-8-12-14-16; 20-22-24-28-30-32;36-38-40-44-46-48; 52-54-56-60-62-64; 68-70-72-76-78-80;84-86-88-92-94-96; 100-102-104-108-110-112; 116-118-120-124-126-128;132-134-136-140-142-144; 148-150-152-156-158-160;164-166-168-172-174-176; 180-182-184-188-190-192;196-198-200-204-206-208; 212-214-216-220-222-224;228-230-232-236-238-240; 244-246-248-252-254-256;260-262-264-268-270-272; 276-278-280-284-286-288;292-294-296-300-302-304; and 310-312-314-318-320-322.
 8. The method ofclaim 4, wherein the antibody or antigen-binding fragment comprises: (a)a heavy chain variable region (HCVR) having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82,98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, and 308;and (b) a light chain variable region (LCVR) having an amino acidsequence selected from the group consisting of SEQ ID NOs: 10, 26, 42,58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282,298, and
 316. 9. The method of claim 1[[4]], wherein the IL-33antagonist is an IL-33 trap comprising a first IL-33 binding domain (D1)attached to a multimerizing domain (M), wherein D1 comprises anIL-33-binding portion of an ST2 protein.
 10. The method of claim 9,wherein the IL-33 trap further comprises one or more additional IL-33binding domains selected from the group consisting of D2, D3 and D4. 11.The method of claim 1, wherein the IL-33 antagonist is an IL-33 trapcomprising an IL-33-binding portion of an ST2 protein, an extracellulardomain of an IL-1RAcP protein, or other IL-33 binding domain.
 12. Themethod of claim 1, wherein the IL-33 antagonist is an IL-33 trapcomprising the amino acid sequence selected from the group consisting ofSEQ ID NOs: 323, 324, 325, 326 and
 335. 13. The method of claim 1,further comprising administering an effective amount of a secondtherapeutic agent useful for diminishing at least one symptom of anIL-33 mediated disease or disorder.
 14. The method of claim 13, whereinthe second therapeutic agent is selected from the group consisting of anon-steroidal anti-inflammatory (NSAID), a corticosteroid, a bronchialdilator, an antihistamine, epinephrine, a decongestant, a thymic stromallymphopoietin (TSLP) antagonist, an IL-13 antagonist, an IL-4antagonist, an IL-4/IL-13 dual antagonist, an IL-5 antagonist, an IL-6antagonist, an IL-12/23 antagonist, an IL-22 antagonist, an IL-25antagonist, an IL-17 antagonist, an IL-31 antagonist, an oral PDE4inhibitor and another IL-33 antagonist or a different antibody orreceptor based antagonist to IL-33.
 15. (canceled)
 16. The method ofclaim 1, wherein the biomarker is calcitonin, and wherein the calcitoninlevel increases in the serum of the subject having an IL-33 mediateddisease or disorder, and wherein the increase in serum calcitonincorrelates with an increased level of calcitonin and IL-33 in the lungsof the subject.
 17. The method of claim 16, wherein the increased levelof calcitonin in the serum of the subject having an IL-33 mediateddisease or disorder is reduced to the reference level followingtreatment with the anti-IL-33 antagonist.
 18. The method of claim 1,wherein the biomarker is calcitonin, the biological sample is a serumsample, and the reference level of calcitonin is 5 pg/mL for a femalesubject and 10 pg/mL for a male subject.
 19. The method of claim 1,wherein the IL-33 mediated disease or disorder is an inflammatorydisease or disorder selected from the group consisting of asthma,allergy, allergic rhinitis, allergic airway inflammation, atopicdermatitis (AD), chronic obstructive pulmonary disease (COPD),inflammatory bowel disease (IBD), multiple sclerosis, arthritis,psoriasis, eosinophilic esophagitis, eosinophilic pneumonia,eosinophilic psoriasis, hypereosinophilic syndrome, graft-versus-hostdisease, uveitis, cardiovascular disease, pain, multiple sclerosis,lupus, vasculitis, chronic idiopathic urticaria and EosinophilicGranulomatosis with Polyangiitis (Churg-Strauss Syndrome). 20-32.(canceled)
 33. The method of claim 1, wherein the biological sample fromthe subject is a solid tissue sample, a cell sample, or a blood sample.34. The method of claim 33, wherein the solid tissue sample is selectedfrom the group consisting of heart, kidney, liver, lung and spleen. 35.The method of claim 33, wherein the cell sample is a dorsal rootganglion cell sample, sputum cell sample, bronchoalveolar lavage cellsample, nasal polyps cell sample, fecal cell sample, lung, colon heart,kidney, skin biopsy, or spleen biopsy cell sample.
 36. The method ofclaim 33, wherein the blood sample is whole blood, plasma, or serum.37-52. (canceled)
 53. The method of claim 8, wherein the antibody orantigen-binding fragment comprises a HCVR/LCVR pair comprising the aminoacid sequences of SEQ ID NO: 274/282.